Gemini virus vectors for gene expression in plants

ABSTRACT

A gene amplification system based on plant viral genetic elements dramatically increases foreign protein production in plants. A safer and more economical production system for vaccines and antibodies in recombinant plants grown using agricultural practice is described. The high-level expression system uses the replicative process of a plant mastrevirus, exemplified by bean yellow dwarf virus (BeYDV). The expression system is preferably inducible to avoid interference with plant growth and development. Developmental cues, such as fruit ripening, are employed to trigger expression of the foreign protein using a tissue-specific promoter. A single, stably integrated expression cassette for foreign protein is replicated extrachromosomally in ripening fruit, forming hundreds of transcriptionally competent copies. Preferred plant hosts include tomato as a model system and soybean for production of large quantities of protein at high total protein levels.

TECHNICAL FIELD

[0001] The present invention is related to genetic engineering ofplants. The invention is particularly related to the transformation ofplants using recombinant DNA techniques to amplify a gene of interestand express a protein of interest.

BACKGROUND OF THE INVENTION

[0002] A predominant mode of plant transformation employs A.tumefaciens, in which a transforming DNA (T-DNA) is modified toincorporate a desired foreign gene. The recombinant T-DNA contains thedesired foreign gene between flanking non-coding regulatory sequencesand the left and right border regions of the wild-type tumor-inducing(Ti) plasmid. The recombinant T-DNA can be provided as part of anintegrative plasmid, which integrates into a wild-type Ti plasmid byhomologous recombination. Typically, however, the recombinant T-DNA isprovided in a binary vector and transferred into a plant cell throughthe action of trans-acting vir genes on a helper Ti plasmid. The T-DNAintegrates randomly into the nuclear genome with some of thetransformants permitting expression of the desired protein (Zambryski,1988). Because of the random integration event of the T-DNA into thenuclear chromosome, variability of transcription level is expected, andtransformants are screened to identify those showing the highest levelsof foreign gene expression. Those transformants expressing the highestlevels of foreign protein can be propagated and multiplied in tissueculture before transplanting to soil.

[0003] Many plant species previously recalcitrant to gene transfer arenow amenable, including cereal crops (McElroy et al., 1994). Plants havethe capacity to express foreign genes from a wide range of sources,including viral, bacterial, fungal, insect, animal, and other plantspecies. In single-copy nuclear transgenics, foreign protein in excessof 1% of total protein is often achieved (Hiatt et al., 1989). Further,assembly and processing of complex animal proteins in plants ispossible, e.g., human serum albumin (Sijmons et al., 1990) and secretoryantibodies (Ma et al., 1995a). Recently, expression of correctlyprocessed avidin was reported in corn seed at a level of 2% of the totalsoluble protein (Hood et al., 1997). It has been estimated that the costof recombinant protein production in plants (assuming the foreignprotein is 10% of total protein) can be 10 to 50 times less than in E.coli by fermentation (Kusnadi et al., 1997).

[0004] Plants have been used as expression systems for vaccine antigens(Mason et al., 1995). The expression of vaccine antigens in tobaccoplants has been reported and the plant material has been shown to beorally immunogenic in mice (work reviewed by Mason et al., 1995; Amtzenet al., 1996). Complex antibodies have also been expressed in plants,which correctly processed and assembled the antibody chains into IgG andsecretory IgA forms (review by Ma et al., 1995b). In the latter case,four different genes were coordinately expressed, including the IgAheavy and light chains, the joining component, and the secretorycomponent, which faithfully assembled in plant cells. Further,expression and accumulation of antibodies in corn and soybean seeds hasbeen reported.

[0005] However, a major limitation in the use of plants for expressionand delivery of a protein of interest is the rather low level ofexpression usually obtained, which ranges from 0.01% to 2% of the totalsoluble protein. For example, soybeans contain 40% protein by weight,yet current methods for foreign protein expression yield no more than 2%of the total protein in seeds. Synthetically produced recombinantvaccine proteins, which avoid the hazards associated with using live orattenuated virus, can be produced in cell culture systems, e.g.,hepatitis B surface antigen (Cregg et al., 1987) and dengue virusproteins (Sugrue et al., 1997). However, the cost of cell culturesystems is often so high as to preclude vaccination on a large scale,particularly in poor countries.

[0006] In applications requiring overexpression of a purified protein ofinterest, high-level expression would greatly facilitate thepurification process. Therefore, a method of amplifying a gene ofinterest and overproducing a protein of interest in recombinant plantsis desired.

[0007] Previous techniques are, however, inherently self-limiting byvirtue of “successful” transformation affording only one or a fewfunctional copies of the foreign gene integrated into the plant genome.Efforts to increase the level of expression under such circumstances aretherefore limited to optimizing the promoter and/or enhancer sequences,using synthetic versions of the foreign gene optimized for expression inthe plant host, optimizing the termination sequence, optimizingexpression of transcription factors, and the like. These measures can beexpected to enhance expression of the desired antigen, although suchenhancement is still limited by the copy number of the foreign gene.True “amplification” of the foreign gene in plant cells, in whichmultiple functional copies of the gene are generated eitherextrachromosomally or integrated into the plant chromosome, is desiredif much greater levels of protein expression are to be achieved. Thegeminiviruses are interesting candidates for producing markedamplification of transgenes in plants.

[0008] Members of the plant virus taxonomic family Geminiviridae areunique among viruses in possessing twinned or geminate virions. They arealso unusual among plant viruses in that they possess single-strandedcircular DNA genomes. The three genera of Geminiviridae are: theleafhopper-transmitted Mastreviruses (type member: maize streak virus,MSV); the leaf- and planthopper-transmitted Curtoviruses (type member:beet curly top virus, BCTV); and the whitefly-transmitted Begomoviruses(type member: bean golden mosaic virus, BGMV). Until recently, the threegenera were known as Subgroups I, II and III, respectively.Mastreviruses and Curtoviruses have only a single genomic component ofapproximately 2.5 to 2.8 kb; Begomoviruses may have one or twocomponents of the same size, one of which is dependent on the other forreplication. Mastreviruses have the simplest organization, withCurtoviruses and Begomoviruses sharing a very similar and more complexorganization. An overview of the genetic organization of geminivirusgenomes is shown in FIG. 1.

[0009] The geminiviruses replicate via a rolling circle mechanism,analogous to that used by phage Φ174 and ssDNA plasmids of gram positivemicroorganisms. The only exogenous protein required for replication isthe viral replication initiation (Rep) protein encoded by a geminiviralreplicase gene. This multifunctional protein initiates replication at aconserved stem loop structure found in the viral origin of replicationby inducing a nick within a conserved nonanucleotide motif (TAATATTA↓C)found in the intergenic loop sequence. Transcription of the viral genomeis bidirectional with transcription initially within the intergenic (IR)region. Rep also has functions involved in controlling the plant cellcycle, and possibly also in modulating the expression of host genesinvolved in DNA replication (reviewed by Palmer et al., 1997b). The Repprotein can act in trans, that is, it need not be expressed by the viralreplicon itself, but can be supplied from another extrachromosomal viralreplicon, or even from a nuclear transgene (Hanley-Bowdoin et al.,1990). The cis requirements for viral replication are the viralintergenic region/s (IR), which contain sequences essential forinitiation of rolling circle replication (the long intergenic region(LIR) of Mastreviruses, or the intergenic region of other geminiviruses)and synthesis of the complementary strand (the short IR (SIR) ofMastreviruses).

[0010] Infectious clones of geminiviruses are commonly constructed astandem dimers or partial dimers of the virus genome, usually with theorigin of replication sequences duplicated. This facilitates escape ofthe cloned virus from the cloning vector sequences by a replicativerelease mechanism mediated by the Rep protein inducing a nick at eachstem-loop structure and the host DNA replication machinery thendisplacing a ssDNA copy of the viral genome. This mechanism applies torescue of replication-defective geminivirus genomes from chromosomallyintegrated partial multimers by the Rep protein of wild type virus (see,for example, Stanley et al., 1990). Moreover, the Rep protein canmediate replicative release of recombinant viral DNA integrated into thehost cell chromosome (Hayes et al., 1988; 1989; Kanevski et al., 1992;Palmer, 1997; Palmer et al., 1997d).

[0011] Geminiviruses replicate to very high copy number in the nuclei ofinfected cells, via a double-stranded DNA replicative intermediate form(RF-DNA) and have therefore attracted interest for their potential usein gene amplification strategies to increase the copy number and enhancethe expression levels of foreign genes linked to the viral replicon(reviewed by Palmer et al., 1997a and Timmermans et al., 1994).Geminiviruses seem to be fairly plastic with respect to the size offoreign DNA that can be linked to the viral replicon without inhibitingDNA replication. There is, however, a stringent size limitation imposedon movement of virus genomes. This issue becomes irrelevant, however, ifthe geminiviral replicon is used in a transgene amplification systemwhere a partial dimer of a “master copy” of the viral replicon (lackingthe genes involved in viral movement) is integrated into every cell of atransgenic plant (Palmer et al., 1997a). The circular monomeric viralreplicon is then mobilized from the master copy in the chromosome by areplicative release mechanism (Stenger et al., 1991), and therecombinant viral vector replicates to very high copy number as anuclear episome (e.g. Hayes et al. ,1988; 1989; Kanevski et al., 1992;Palmer et al., 1997d). The RF-DNA forms of geminiviral genomes exist ashistone-associated minichromosome structures, and their genes aretranscribed by the host RNA polymerase complex. Transcription of geneslinked to a geminiviral replicon should therefore be regulated by theirrespective promoter sequences.

[0012] U.S. Pat. No. 5,589,379 and 5,650,303, issued to Kridl et al.,disclose use of a geminiviral gene transfer vector to permit inducibleexpression of a foreign gene in plants. In this approach, a firstexpression cassette has a foreign protein coding sequence under thecontrol of the coat protein promoter native to the geminivirus. A secondexpression cassette has a geminiviral trans-acting transcription factorunder the control of a plant-inducible promoter. In this way,transcription of the foreign gene reportedly can be regulated by theinducible expression of the trans-acting transcription factor.

[0013] A gene amplification approach to increasing the copy number andexpression of recombinant transgenes could dramatically increase thelevel of desired foreign proteins in plants. This could lead to saferand more economical production of a protein of interest. In this regard,a transgene vector system that utilizes the release and replicationcapabilities of geminiviruses is particularly worthy of investigation.

[0014] Needham et al. disclose a binary vector containing the followingelements derived from tobacco yellow dwarf virus: two copies of an LIRflanking an SIR and two complementary sense open reading frames (C1 andC2) which contain an intron, which when processed, produces a Repprotein under the transcriptional control of its native promoter withinthe LIR. This vector does not include open reading frames encoding theputative movement (V1) and coat (V2) proteins. Needham et al. disclosefurther that transgenic tobacco plants transformed with this vectorfurther comprising a reporter gene, demonstrate release and episomalreplication of the viral elements of the vector, and expression of thereporter gene (Needham et al., 1998, Plant Cell Report, 17:631).

[0015] Atkinson et al. disclose an episomal vector containing thefollowing elements derived from tobacco yellow dwarf virus: two copiesof an LIR flanking both an SIR and two complementary sense open readingframes, C1 and C2 that produce Rep. The expression of the Rep protein isunder the transcriptional control of its native promoter within the LIR.Atkinson et al. also disclose that transgenic Petunia hybrida plantscontaining a CaMv 35S promoter-riven chalcone synthase A gene clonedinto the episomal vector, demonstrate release and pisomal replication ofthe viral elements of the vector and the chalcone synthase A gene (Rosst al., 1998, The Plant Journal, 15:593).

[0016] There is a need in the art for a method of increasing the copynumber of a recombinant transgene in a plant.

[0017] There is also a need in the art for a method of increasing thelevel of expression of a recombinant transgene in a plant.

[0018] There is also a need in the art for a method of increasing thelevel of protein expressed from a recombinant transgene in a plant.

SUMMARY OF THE INVENTION

[0019] The invention provides a pair of recombinant nucleic acidmolecules wherein a first molecule comprises at least a portion of along intergenic region (LIR) of a geminivirus genome and wherein thefirst molecule lacks a functional geminiviral coat protein encodingsequence, and a second molecule comprising a geminiviral replicase geneoperably linked to a fruit ripening-dependent promoter.

[0020] As used herein, a “long intergenic region” (LIR) refers to anoncoding region that contains sequences capable of forming a hairpinstructure, including a conserved 9-base sequence (TAATATTA↓C) found inall geminiviruses.

[0021] As used herein, “at least a portion of a long intergenic region”refers to a region of a long intergenic region (LIR) that contains a repbinding site capable of mediating excision and replication by ageminivirus Rep protein. The LIR of the geminivirus, bean yellow dwarfvirus is 303 nucleotides (FIG. 2). As used herein, “at least a portionof a long intergenic repeat” refers to a fragment of the long intergenicrepeat that is less than 303 nucleotides. “At least a portion” of a longintergenic region encompasses for example 50 nucleotides, 100nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 264nucleotides, 270 nucleotides, 275 nucleotides, 280 nucleotides, 285nucleotides, 290 nucleotides or 300 nucleotides. An LIR according to theinvention includes an LIR of mastrevirus as well as an intergenic region(IR) of either curtovirus or begomovirus.

[0022] As used herein, “fruit ripening dependent” refers to inducibleunder fruit ripening conditions and/or expressed in a tissue specificmanner.

[0023] By “tissue specific” is meant expressed only in tissues of thefruit or in leaves. Tissues of the fruit include vascular bundles,pericarp, collumella, epidermis, placental tissue, locular tissue andseeds. As used herein, “tissue specific” also refers to expressed in aseed specific manner.

[0024] As used herein, “seed specific” refers to expressed only in thedeveloping seed encompassing the cotyledon and the embryo axis, but notincluding the root and the stem. As used herein, “seed specific” alsorefers to expressed in the endosperm and perisperm.

[0025] A “fruit ripening-dependent promoter” according to the inventiondoes not include a native viral Rep protein promoter included in an LIR.The LIR sequence of Bean yellow dwarf virus, including the putative TATAbox (TTATA, boxed in FIG. 2) is presented in FIG. 2, for example.Sequences of other geminivirus LIRs are available in the literature. Asused herein, the native rep gene promoter is located in the LIR and canregulate rep gene expression in a phloem specific manner. Therefore, theinvention does not encompass control of the rep gene by a native reppromoter, which native promoter is found in an LIR (FIG. 2), andincludes the TATA sequence TTATA (boxed in FIG. 2).

[0026] As used herein, a “fruit ripening-dependent promoter” can also beexpressed in phloem wherein it demonstrates development-stage dependentexpression encompassing expression during fruit ripening or embryostorage protein deposition.

[0027] As used herein, “development stage” refers to a particular periodof cell growth, differentiation, and organization of cells which can becharacterized by changes in anatomy, biochemistry, or the coordinateexpression of a particular set of genes.

[0028] As used herein, “developing” refers to the process of growth,differentiation and organization of cells that occurs during theformation of a tissue (e.g. epidermis, phloem, xylem, parenchyma etc . .. ) or an organ (e.g. leaf, stem, root, flower, fruit or seed).

[0029] As used herein, “fruit” refers to the ovary of an angiospermflower and the associated structures (e.g. the receptacle or parts ofthe floral tube) that enlarge and develop to form a mass of tissuesurrounding the seeds. According to the invention, the particulartissues that are involved in fruit development vary with the species,but tissues involved in fruit development according to the invention,are always derived from the maternal parent of the progeny seeds.

[0030] As used herein, “ripe” refers to a stage of fruit developmentthat is characterized by changes in pigmentation, the conversion ofacids and starches to free sugars, and breakdown of cell walls thatresults in softening of the fruit.

[0031] As used herein, “fruit ripening conditions” refer to conditionsunder which the developmental processes involved in fruit ripening canoccur, including cell division and expansion of maternal tissues thatoccurs after fertilization of ovaries. As used herein, for example,production of ethylene is a chemical signal that stimulates the geneticprogram for ripening in climacteric fruits such as tomato.

[0032] As used herein, “embryo storage protein deposition” refers to thesynthesis and accumulation of storage proteins in the parts of theembryo, particularly cotyledons, or seeds of species that lack endospermand in which the embryo is large and contains most of the seed storagetissue (including for example, Leguminosae, Cucurbitaceae, Compositaea,Solanaceae, Brassicaceae).

[0033] “Inducible” refers to expressed in the presence of an exogenousor endogenous chemical (for example an alcohol, a hormone, or a growthfactor), in the presence of light and/or in response to developmentalchanges.

[0034] As used herein, “endogenous” refers to naturally occurring in aplant.

[0035] As used herein, “exogenous” refers to not naturally occurring ina plant.

[0036] As used herein, “inducible” also refers to expressed in anytissue in the presence of a chemical inducer”. As used herein, “chemicalinduction” according to the invention refers to the physical applicationof a exogenous or endogenous substance (including macromolecules e.g.proteins, or nucleic acids) to a plant or a plant organ (e.g. byspraying a liquid solution comprising a chemical inducer on leaves,application of a liquid solution to roots or exposing plants or plantorgans to gas or vapor) which has the effect of causing the targetpromoter present in the cells of the plant or plant organ to increasethe rate of transcription.

[0037] As used herein, “protein of interest” refers to any protein thatis either heterologous or endogenous to a geminivirus or plant.

[0038] As used herein, “gene of interest” refers to any gene that iseither heterologous or endogenous to a geminivirus or plant.

[0039] As used herein, “nucleotide sequence of interest” refers to anynucleotide sequence that is either heterologous or endogenous to ageminivirus or plant. A nucleotide sequence of interest refers to DNA orRNA.

[0040] “Heterologous” refers to a gene which is not naturally present ina geminivirus genome or a gene which is not naturally present in a plantgenome. “Heterologous” also refers to a protein which is not naturallyexpressed from a geminivirus genome or a plant genome.

[0041] “Endogenous” refers to a gene which is naturally present in ageminivirus genome or a plant genome. “Endogenous” also refers to aprotein which is naturally expressed from a geminivirus genome or aplant genome.

[0042] As used herein, the term “operably linked” refers to therespective coding sequence being fused in-frame to a promoter, enhancer,termination sequence, and the like, so that the coding sequence isfaithfully transcribed, spliced, and translated, and the otherstructural features are able to perform their respective functions.

[0043] In a preferred embodiment, the first molecule further comprisesan SIR.

[0044] As used herein, “SIR” refers to a noncoding region of aMastrevirus genome containing the putative complementary strand originof replication, the binding site for a short DNA primer that primessynthesis of the complementary DNA strand and consensus polyadenylationsignals in both strands. As used herein, “SIR” refers to a region of DNAthat is approximately 150 base pairs and extends from the terminationcodon of the geminivirus coat proteins (V2) to the termination codon ofone of the open reading frames encoding the Rep protein (C2).

[0045] According to the invention, a gene of interest can be locatedeither 5′ of an SIR or 3′ of an SIR in a recombinant nucleic acidmolecule.

[0046] In another preferred embodiment, the first molecule furthercomprises a plant-functional promoter.

[0047] In another preferred embodiment, the plant-functional promoter isselected from the group consisting of CaMV 35S, tomato E8, patatin,ubiquitin, mannopine synthase (mas), rice actin 1. soybean seed proteinglycinin (Gy1) and soybean vegetative storage protein (vsp).

[0048] In another preferred embodiment, the first molecule furthercomprises a gene of interest.

[0049] In another preferred embodiment, the gene of interest is aheterologous gene.

[0050] In another preferred embodiment, the gene of interest of thefirst molecule is selected from the group consisting of a gene encodingluciferase, glucuronosidase (GUS), green fluorescent protein (GFP),shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), E. coli labiletoxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis Bsurface antigen (HBsAg).

[0051] In another preferred embodiment, the first molecule furthercomprises a plant-functional termination sequence.

[0052] In another preferred embodiment, the plant-functional terminationsequence is selected from the group consisting of nopaline synthase(nos), vegetative storage protein (vsp), pin2, and geminiviral shortintergenic (sir) termination sequences.

[0053] In another preferred embodiment, the nucleotide sequence of thefirst DNA molecule is optimized for expression in plants by having atleast one codon degenerate to a corresponding codon of the nativeprotein encoding sequence.

[0054] In another preferred embodiment, the first molecule is singlestranded.

[0055] The invention also provides a recombinant nucleic acid moleculecomprising at least a portion of a long intergenic region (LIR) of ageminivirus genome and a geminiviral replicase gene operably linked to afruit ripening-dependent promoter.

[0056] In a preferred embodiment, the recombinant nucleic acid moleculefurther comprises an SIR.

[0057] In another preferred embodiment, the recombinant nucleic acidmolecule further comprises a plant-functional promoter.

[0058] In another preferred embodiment, the plant-functional promoter isselected from the group consisting of CaMV 35S, tomato E8, patatin,ubiquitin, mannopine synthase (mas), rice actin 1, soybean seed proteinglycinin (Gy1) and soybean vegetative storage protein (vsp).

[0059] In another preferred embodiment, the recombinant nucleic acidmolecule further comprises a gene of interest.

[0060] In another preferred embodiment, the gene is a heterologous gene.

[0061] In another preferred embodiment, the gene of interest is selectedfrom the group consisting of a gene encoding luciferase, glucuronosidase(GUS), green fluorescent protein (GFP), shigatoxin B (StxB),staphylococcus enterotoxin B (SEB), E. coli labile toxin B (LT-B),Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen(HbsAg).

[0062] In another preferred embodiment, the recombinant nucleic acidmolecule further comprises a plant-functional termination sequence.

[0063] In another preferred embodiment, the plant-functional terminationsequence is selected from the group consisting of nopaline synthase(nos), vegetative storage protein (vsp), pin2, and geminiviral shortintergenic (sir) termination sequences.

[0064] In another preferred embodiment, the nucleotide sequence isoptimized for expression in plants by having at least one codondegenerate to a corresponding codon of the native protein encodingsequence.

[0065] In another preferred embodiment, the recombinant nucleic acidmolecule is single stranded.

[0066] The invention also provides an expression vector comprising aselectable marker gene and at least a portion of a long intergenicregion (LIR) of a geminivirus genome, a restriction site for insertionof a gene of interest, a functional geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter, and wherein said nucleicacid sequence lacks a functional geminiviral coat protein encodingsequence.

[0067] In a preferred embodiment, the vector further comprises an SIR.

[0068] In another preferred embodiment, the vector lacks a functionalgeminiviral replicase gene.

[0069] In another preferred embodiment, the nucleotide sequence isflanked by two of said LIR portions.

[0070] In another preferred embodiment, the 5′ end of the nucleotidesequence is operably linked to a plant-functional promoter sequence.

[0071] In another preferred embodiment, the vector further comprises agene of interest.

[0072] In another preferred embodiment, the gene is a heterologous gene.

[0073] In another preferred embodiment, the gene of interest is selectedfrom the group consisting of a gene encoding a luciferase,glucuronosidase (GUS), green fluorescent protein (GFP), shigatoxin B(StxB), staphylococcus enterotoxin B (SEB), labile toxin B (LT-B),Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen(HbsAg).

[0074] In another preferred embodiment, the 3′ end of the gene isoperably linked to a plant-functional termination sequence.

[0075] In another preferred embodiment, the gene is optimized forexpression in plants by having at least one codon degenerate to acorresponding codon of the native protein encoding sequence.

[0076] In another preferred embodiment, the vector further comprises anE. coli origin of replication.

[0077] In another preferred embodiment, the vector further comprises anAgrobacterium tumefaciens origin of replication.

[0078] In another preferred embodiment, the nucleotide sequence isflanked by left and right T-DNA border regions of Agrobacteriumtumefaciens.

[0079] The invention also provides for a strain of E. coli transfectedwith an expression vector comprising a selectable marker gene and atleast a portion of a long intergenic region (LIR) of a geminivirusgenome, a restriction site for insertion of a gene of interest, afunctional geminiviral replicase gene operably linked to a fruitripening-dependent promoter, and wherein said nucleic acid sequencelacks a functional geminiviral coat protein encoding sequence, andfurther comprising an E. coli origin of replication.

[0080] The invention also provides for a strain of Agrobacteriumtumefaciens transfected with an expression vector comprising aselectable marker gene and at least a portion of a long intergenicregion (LIR) of a geminivirus genome, a restriction site for insertionof a gene of interest, a functional geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter, and wherein said nucleicacid sequence lacks a functional geminiviral coat protein encodingsequence, and further comprising an E. coli origin of replication,further comprising an Agrobacterium tumefaciens origin of replication.

[0081] In a preferred embodiment, the strain further comprises a helpertumor-inducing (Ti) plasmid.

[0082] The invention also provides for a transgenic plant celltransformed with a nucleic acid having at least a portion of a longintergenic region (LIR) of a geminivirus genome, a gene of interest,wherein the nucleic acid lacks a functional geminiviral coat proteinencoding sequence.

[0083] The invention also provides for a transgenic plant celltransformed with a nucleic acid comprising at least a portion of a longintergenic region (LIR) of a geminivirus genome flanking a restrictionsite for insertion of a gene of interest, and a functional geminiviralreplicase gene operably linked to a fruit ripening-dependent promoterand wherein the nucleic acid sequence lacks a functional geminiviralcoat protein encoding sequence.

[0084] In a preferred embodiment, the transgenic plant cell furthercomprises a heterologous gene.

[0085] In another preferred embodiment, the transgenic plant cell lacksa functional geminiviral replicase gene.

[0086] In another preferred embodiment, the nucleic acid is present innuclear episomes in the cell.

[0087] In another preferred embodiment, the 5′ end of the gene ofinterest is operably linked to a plant-functional promoter sequence.

[0088] In another preferred embodiment, the gene of interest is selectedfrom the group consisting of a gene encoding luciferase, glucuronosidase(GUS), green fluorescent protein (GFP), shigatoxin B (StxB),staphylococcus enterotoxin B (SEB), labile toxin B (LT-B), Norwalk viruscapsid protein (NVCP), and hepatitis B surface antigen (HBsAg).

[0089] In another preferred embodiment, the 3′ end of the gene ofinterest is operably linked to a plant-functional termination sequence.

[0090] In another preferred embodiment, the gene of interest isoptimized for expression in plants by having at least one codondegenerate to a corresponding codon of the native protein encodingsequence.

[0091] In another preferred embodiment, the transgenic plant cellfurther comprises a viral replicase encoding sequence operably linked toa plant functional promoter and a termination sequence.

[0092] In another preferred embodiment, transcription of the viralreplicase encoding sequence is regulated by an inducible promoter.

[0093] In another preferred embodiment, the 5′ end of the viralreplicase encoding sequence is operably linked to a tissue-specificpromoter.

[0094] In another preferred embodiment, the tissue-specific promoter isselected from the group consisting of glucocorticoid, estrogen, jasmonicacid, insecticide RH5992, copper, tetracycline, and alcohol-induciblepromoters.

[0095] In another preferred embodiment, the viral replicase encodin,sequence encodes a wild-type geminiviral replicase.

[0096] In another preferred embodiment, the viral replicase encodingsequence is provided as an expression cassette or viral replicon.

[0097] The invention also provides for a transgenic plant seedtransformed with a nucleic acid having at least a portion of a longintergenic region (LIR) of a geminivirus genome, a gene of interest,wherein the nucleic acid lacks a functional geminiviral coat proteinencoding sequence.

[0098] The invention also provides for a transgenic plant seedtransformed with a nucleic acid comprising at least a portion of a longintergenic region (LIR) of a geminivirus genome, a restriction site forinsertion of a gene of interest, and a functional geminiviral replicasegene operably linked to a fruit ripening-dependent promoter and whereinthe nucleic acid sequence lacks a functional geminiviral coat proteinencoding sequence.

[0099] In a preferred embodiment, the seed further comprises aheterologous gene.

[0100] In another preferred embodiment, the nucleic acid lacks afunctional geminiviral replicase gene.

[0101] In another preferred embodiment, the seed further comprises aviral replicase encoding sequence expressed in trans with the nucleotidesequence.

[0102] In another preferred embodiment, the 5′ end of the viralreplicase encoding sequence is operably linked to a fruitripening-dependent promoter.

[0103] In another preferred embodiment, the seed is selected fromtobacco, tomato, potato, banana, soybean, pepper, wheat, rye, rice,spinach, carrot, maize and corn.

[0104] The invention also provides for a method of transforming a plantcell comprising contacting the plant cell with a strain of Agrobacteriumtumefaciens transfected with an expression vector comprising aselectable marker gene and a nucleic acid sequence comprising at least aportion of a long intergenic region (LIR) of a geminivirus genome, arestriction site for insertion of a gene of interest, and a functionalgeminiviral replicase gene operably linked to a fruit ripening-dependentpromoter and wherein said nucleic acid sequence lacks a functionalgeminiviral coat protein encoding sequence, and further comprising anAgrobacterium tumefaciens origin of replication, under conditionseffective to transfer and integrate the nucleotide sequence into thenuclear genome of the cell.

[0105] In a preferred embodiment, the transformed plant cell isregenerated.

[0106] The invention also provides a method of transforming a plant cellcomprising subjecting the plant cell to microparticle bombardment withsolid particles loaded with a pair of recombinant nucleic acid moleculeswherein a first molecule comprises at least a portion of a longintergenic region (LIR) of a geminivirus genome, and wherein the firstmolecule lacks a functional geminiviral coat protein encoding sequence,and a second molecule comprising a geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter, under conditionseffective to transfer and integrate said nucleotide sequence into thenuclear genome of the cell.

[0107] The invention also provides a method of producing a transgenicplant comprising transforming a plant cell by a method comprisingsubjecting the plant cell to microparticle bombardment with solidparticles loaded with a pair of recombinant nucleic acid moleculeswherein a first molecule comprises at least a portion of a longintergenic region (LIR) of a geminivirus genome, and wherein the firstmolecule lacks a functional geminiviral coat protein encoding sequence,and a second molecule comprising a geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter under conditions effectiveto transfer and integrate said nucleotide sequence into the nucleargenome of the cell, and regenerating the plant cell.

[0108] The invention also provides a method of amplifying a heterologousnucleotide sequence in a transgenic plant comprising producing atransgenic plant by a method comprising transforming a plant cell by amethod comprising subjecting the plant cell to microparticle bombardmentwith solid particles loaded with a pair of recombinant nucleic acidmolecules wherein a first molecule comprises at least a portion of along intergenic region (LIR) of a geminivirus genome, and wherein thefirst molecule lacks a functional geminiviral coat protein encodingsequence, and a second molecule comprising a geminiviral replicase geneoperably linked to a fruit ripening-dependent promoter under conditionseffective to transfer and integrate said nucleotide sequence into thenuclear genome of the cell, and regenerating the plant cell, andsubjecting the transgenic plant to a wild-type geminivirus, whichexpresses a viral replicase in planta that rescues and replicates thenucleotide sequence in cells of the plant.

[0109] The invention also provides a method of overproducing a proteinin a plant comprising producing a transgenic plant by the method ofcontacting the plant cell with a strain of Agrobacterium tumefacienstransfected with an expression vector comprising a selectable markergene and a nucleic acid sequence comprising at least a portion of a longintergenic region (LIR) of a geminivirus genome, a restriction site forinsertion of a gene of interest, and a functional geminiviral replicasegene operably linked to a fruit ripening-dependent promoter and whereinsaid nucleic acid sequence lacks a functional geminiviral coat proteinencoding sequence, and further comprising an Agrobacterium tumefaciensorigin of replication, under conditions effective to transfer andintegrate the nucleotide sequence into the nuclear genome of the cell,ans subjecting the transgenic plant to a wild-type geminivirus, whichexpresses a viral replicase in planta that rescues and replicates thenucleotide sequence in said plant.

[0110] The invention also provides for a method of amplifying aheterologous nucleotide sequence in a transgenic plant comprisingproducing a transgenic plant by the method of contacting the plant cellwith a strain of Agrobacterium tumefaciens transfected with anexpression vector comprising a selectable marker gene and a nucleic acidsequence comprising at least a portion of a long intergenic region (LIR)of a geminivirus genome, a restriction site for insertion of a gene ofinterest, and a functional geminiviral replicase gene operably linked toa fruit ripening-dependent promoter and wherein said nucleic acidsequence lacks a functional geminiviral coat protein encoding sequence,and further comprising an Agrobacterium tumefaciens origin ofreplication, under conditions effective to transfer and integrate thenucleotide sequence into the nuclear genome of the cell, and subjectingthe transgenic plant to a chemical or developmental agent, which inducesexpression of a viral replicase in planta that rescues and replicatesthe nucleotide sequence in the plant.

[0111] In a preferred embodiment, the inducible promoter is selectedfrom the group consisting of glucocorticoid, estrogen, andalcohol-inducible promoters.

[0112] In another preferred embodiment, replication of the viralreplicase is expressed in trans with the nucleotide sequence.

[0113] The invention also provides for a method of overproducing aprotein in a plant, comprising producing a transgenic plant produced bythe method comprising contacting the plant cell with a strain ofAgrobacterium tumefaciens transfected with an expression vectorcomprising a selectable marker gene and a nucleic acid sequencecomprising at least a portion of a long intergenic region (LIR) of ageminivirus genome, a restriction site for insertion of a gene ofinterest, and a functional geminiviral replicase gene operably linked toa fruit ripening-dependent promoter and wherein said nucleic acidsequence lacks a functional geminiviral coat protein encoding sequence,and further comprising an Agrobacterium tumefaciens origin ofreplication, under conditions effective to transfer and integrate thenucleotide sequence into the nuclear genome of the cell, and subjectingthe transgenic plant to a chemical or developmental agent, which inducesexpression of a viral replicase in plants that rescues and replicatesthe nucleotide sequence in the plant.

[0114] The invention also provides a recombinant nucleic acid moleculecomprising a functional geminiviral replicase gene operably linked to afruit ripening-dependent promoter.

[0115] The invention also provides a vector comprising a functionalgeminiviral replicase gene operably linked to a fruit ripening-dependentpromoter.

[0116] The invention also provides a transgenic plant cell transformedwith a nucleic acid comprising a functional geminiviral replicase geneoperably linked to a fruit ripening-dependent promoter.

[0117] In a preferred embodiment, the transgenic plant cell is selectedfrom the group consisting of tobacco, tomato, potato, banana, soybean,pepper, wheat, rye, rice, spinach, carrot, maize and corn.

[0118] The invention also provides a transgenic plant seed transformedwith a nucleic acid comprising a functional geminiviral replicase geneoperably linked to a fruit ripening-dependent promoter.

[0119] In a preferred embodiment, the transgenic plant cell is selectedfrom the group consisting of tobacco, tomato, potato, banana, soybean,pepper, wheat, rye, rice, spinach, carrot, maize and corn.

[0120] The present invention thereby affords a method of amplifying,i.e., increasing the copy number, of a desired nucleotide sequence, inthe genome of a transgenic plant, thereby permitting expression of theencoded protein over basal levels obtained in the absence ofamplification. Moreover, protein expression can be regulated in a fruitripening-dependent manner.

BRIEF DESCRIPTION OF THE DRAWINGS

[0121]FIG. 1 illustrates the genome organization of the geminiviruses.

[0122]FIG. 2 shows the nucleic acid sequence of the LIR of Bean YellowDwarf Virus. Translation start sites for the Rep/RepA protein (C1) andthe movement protein (V1) are marked by arrows. Putative TATA boxes areboxed. Stem and loop are indicated by underlined and outlined textrespectively. Putative introns appear in bold faced letters.

[0123]FIG. 3A shows a replicon construct, whereby a partial dimer can beintegrated into the chromosome of a transgenic plant. Due to a mutationin the Rep protein, it is unable to replicate until Rep protein issupplied. FIG. 3B shows a Rep expression cassette in which the rep gene(C1/C2) is placed under the control of a fruit ripening-dependentpromoter (e.g., tomato E8 promoter or soybean seed protein promoter).FIG. 3C shows a replicon construct whereby a partial dimer can beintegrated into the chromosome of a transgene plant comprising a gene ofinterest and a rep gene under the control of a fruit ripening-dependentpromoter.

[0124]FIG. 4 illustrates the Rep-mediated rescue and replication of achromosome-integrated viral replicon as a high copy number nuclearepisome.

[0125]FIG. 5 illustrates plasmid maps of pBY002, pBY017, pBY019, pBY020,pBY024, pBY027 and pBY028.

[0126]FIG. 6 is a plasmid map of pBY217.

[0127]FIG. 7 is a plasmid map of pBY027.

[0128]FIG. 8 is a plasmid map of pBY028.

[0129]FIG. 9 is a plasmid map of pBY029.

[0130]FIG. 10 is the nucleic acid sequence of a rep gene including theintron.

[0131]FIG. 11 is a plasmid map of p35S-Rep.

[0132]FIG. 12 is a plasmid map of p2S-Rep.

[0133]FIG. 13 is a plasmid map of pE8-Rep.

[0134]FIG. 14 is a plasmid map of pBY034.

[0135]FIG. 15 is a plasmid map of pBY036.

[0136]FIG. 16 is a plasmid map of pK!S-ARP(F).

[0137]FIG. 17 is a plasmid map of SRN-ARP(F).

[0138]FIG. 18 is the nucleic acid sequence of a rep gene without theintron.

[0139]FIG. 19 is a plasmid map of p35S-Δintron.

[0140]FIG. 20 is a plasmid map of p2SΔintron.

[0141]FIG. 21 is a plasmid map of pESΔintron.

[0142]FIG. 22 is a plasmid map of pBY035.

[0143]FIG. 23 is a plasmid map of pSRN-ADP(F).

[0144]FIG. 24 is a plasmid map of pBY023.

[0145]FIG. 25 is a plasmid map of pBY032.

[0146]FIG. 26 illustrates BeYDV constructs.

[0147]FIG. 27 is a graph illustrating the time course of GUS expressionin NT1 cells.

[0148]FIG. 28 is a plasmid map of pBYSEB110.

[0149]FIG. 29 is a plasmid map of pBYSEB410.

[0150]FIG. 30 is a plasmid map of pBY031.

[0151]FIG. 31 is a graph illustrating enhancement of GUS expression intobacco NT1 cells.

[0152]FIG. 32A illustrates the strategy for divergent PCR.

[0153]FIG. 32B is gel demonstrating PCR products of a divergent PCRreaction.

[0154]FIG. 33 is a plasmid map of pLIR-GUS.

[0155]FIG. 34 is a plasmid map of pLIR-GUS-SIR.

[0156]FIG. 35 is a plasmid map of pmBY002-BAR.

[0157]FIG. 36 is the nucleotide sequence of a plant-optimized mutantstaphylococcal enterotoxin B (SEB-F44S) gene.

DETAILED DESCRIPTION OF THE INVENTION

[0158] The invention is based on the discovery that DNA constructscomprising components of the viral genome can be excised and episomallyreplicated in the presence of a geminiviral replicase gene encoding aRep protein. These constructs can be used to amplify a gene of interestand to express a protein of interest in a plant. Further, theseconstructs can be used to amplify a gene of interest and express aprotein of interest in a regulated manner in plants in the presence ofgeminiviral replicase gene that is transcriptionally regulated by afruit ripening-dependent promoter.

[0159] As used herein, “episomally replicated” refers to independentreplication of an episome. An “episome” according to the inventionrefers to a mobile genetic element or plasmid that can exist either inan autonomous extrachromosomal state or can be integrated into achromosome”.

[0160] I. Geminiviruses

[0161] Geminiviruses useful according to the invention includeBegomoviruses, Curtoviruses and Mastreviruses.

[0162]FIG. 1 shows the consensus genome organization of double strandedreplicative forms of geminiviruses. Grey boxes indicate the intergenicregions (IR) which contain the origin of replication and transcriptionregulatory regions for bidirectional transcription. The part of theintergenic region which is identical in both Begomovirus genomecomponents is called the common region (CR); as Mastreviruses have twointergenic regions, the region which contains the origin of replicationand viral gene promoter sequences is called the long intergenic region(LIR). The putative complementary strand origin of replication inMastreviruses is in the short intergenic region (SIR), where a short DNAprimer binds, and possibly primes synthesis of the complementary DNAstrand. Open reading frames are shown by arrows, which indicate thedirection in which the ORF is transcribed. Where a gene's function isknown, the name of the gene product is indicated. CP: coat protein; MP:movement protein; Rep: replication initiator protein; TrAP:transcriptional activator protein for virion-sense genes; REn:replication enhancer protein. In Curtoviruses, the C2 ORF does not seemto have transcriptional activator activity. An AV1 ORF is indicated asthe coat protein gene in all cases, whether or not an AV1 ORF ispresent. The position of an intron which results in fusion of theMastrevirus RepA and RepB open reading frames in processed mRNAs isindicated.

[0163] A. Mastreviruses

[0164] Mastreviruses have the simplest genetic organization of anymembers of the plant virus taxonomic family Geminiviridae.

[0165] Species in the Subgroup I (Mastrevirus) genus of thegeminiviruses that are useful according to the invention include beanyellow dwarf virus (BeYDV) (Liu et al., 1997, J. Gen. Virol., 78:2113),Bromus striate mosaic virus (BrSMV), Chloris striate mosaic virus (CSMV)(GenBank M20021), Digitaria streak virus (DSV) (GenBank M23022),Digitaria striate mosaic virus (DiSMV), maize streak virus (MSV)(GenBank AF003952), Miscanthus streak virus, strain natal (MiSV) (DBBJD00800), Panicum streak virus (PanSV-Kar) (GenBank L39638), Paspalumstriate mosaic virus (PSMV), sugarcane streak virus (SSVN)(GenBankM82918), tobacco yellow dwarf virus (TYDV) (GenBank M81103), andwheat dwarf virus (WDV) (EMBL X82104). Tentative species in the genusinclude bajra streak virus (BaSV) and chickpea chlorotic dwarf virus(CpCDV).

[0166] Exemplary of a Mastrevirus vector for use in the presentinvention is bean yellow dwarf virus (BeYDV). BeYDV has only one genomecomponent (Liu et al 1997). It has only three genes, which encode thereplication initiator protein (rep), the movement protein, and the coatprotein (FIG. 1). All of the viral genes are essential for viralinfectivity, but only the rep gene is required for replication (seePalmer & Rybicki, 1997b for a review of Subgroup I geminivirus molecularbiology). BeYDV is preferred for several reasons: as a Mastrevirus,BeYDV has a fairly simple genomic organization; it also has a broad hostrange which encompasses both legumes and solanaceous plants. BeYDV canbe used as a system for enhancing tissue specific gene expression intomatoes or for seed specific expression in soybeans.

[0167] Tobacco yellow dwarf virus (TYDV) (GenBank M81103) is alsoexemplary of a Mastrevirus genome useful according to the invention.

[0168] B. Curtovirus

[0169] Species in the Subgroup II (Curtovirus) genus of thegeminiviruses that are useful according to the invention include but arenot limited to beet curly top virus (BCTV) (GenBank M24597), horseradishcurly top virus (HrCTV) (GenBank U49907) and tomato pseudo curly topvirus (TPCTV) (EMBL X84735).

[0170] C. Begomovirus

[0171] Species in the Subgroup III (Begomovirus) genus of thegeminiviruses that are useful according to the invention include but arenot limited to mungbean yellow mosaic virus (MYMV) (DDBJ D14703), tomatoyellow leaf curl virus strain Israel (TYLCV-Is) (EMBL X15656), Africancassaya mosaic virus strain West Kenya (ACMV-K) (EMBL Z24758), Indiancassaya mosaic virus (ICMV) (EMBL Z24758), Indian tomato leaf curl virus(ItnLCV) (GenBank Z48182), Ageratum yellow vein virus (AgYVV) (EMBLX74516), pepper huasteco virus (PHV) (EMBLX70418), Texas pepper virusstrain Tamaulipas (TPV) (GenBank U57457), tomato golden mosaic virus(TGMV) (GenBank K02029), bean golden mosaic virus (BGMV) (GenBankM88686), potato yellow mosaic virus (PYMV) (DDBJ D00940), bean dwarfmosaic virus (BDMV) (GenBank M88179), tomato mottle virus (TmoV)(GenBank L14460) and Abutilon mosaic virus (AbMV) (EMBL X15983).

[0172] In the present invention, a geminiviral Rep protein can mediatereplication of viral DNA in trans with the appropriate cis-actingsequences providing the viral origin of replication. In one aspect, thedual expression cassettes for a gene of interest, represented by the GUSreporter gene and a rep gene under the transcriptional control of afruit ripening-dependent promoter, are shown illustrated in FIGS. 3A and3B. The expression cassette for a gene of interest either lacks a repgene or contains an inactivated rep gene. Alternatively, these cassettescan be provided together in a single vector, wherein the Rep protein isunder the transcriptional control of a fruit ripening-dependent promoteras illustrated in FIG. 3C. Following transformation, the cassettes canbe present in the plant cell either integrated into the nuclear genomeor extrachromosomal, e.g., as episomes.

[0173] Rescue and replication of a viral cassette wherein the rep geneis inactivated integrated into a plant genome is illustrated in FIG. 4.The trans-acting Rep protein also present within the cell, by virtue ofendogenous production or separate transformation, is shown acting at thecleaved LIR borders of the viral construct so as to release theconstruct from the chromosome.

[0174] II. Constructs Useful According to the Invention

[0175] The invention provides for DNA constructs comprising componentsof the viral genome, wherein a nucleotide sequence encoding a protein ofinterest can be introduced into the DNA construct, and wherein the viralelements of the construct and the nucleotide sequence encoding a proteinof interest can be rescued and episomally replicated in the presence ofa replicase gene under the transcriptional control of a fruitripening-dependent promoter.

[0176] The invention provides for constructs comprising at least aportion of a geminiviral LIR. A portion of the LIR that is usefulaccording to the invention will include enough of the LIR to present thenick recognition sequence of a replicase protein, which permits rescueand replication of the DNA construct from a nuclear genome. A constructuseful according to the invention lacks a functional geminiviral coatprotein encoding sequence so that capsid formation and encapsidation ofthe viral DNA does not occur. A construct that lacks a functionalgeminiviral coat protein encoding sequence can be provided by deleting aportion of the coat protein encoding sequence and replacing the deletedsection with the nucleotide sequence of interest encoding a protein ofinterest. The construct can also be provided as a full or partial dimer,which affords a second LIR recognition sequence and facilitates rescueof the desired sequence.

[0177] Constructs comprising at least a portion of a geminiviral LIR canbe randomly integrated into a chromosome and can be excised by a Repprotein in a site specific manner.

[0178] A preferred geminivirus genome for use as a template, i.e., forperforming mutations and modifications, is that of the mastrevirus beanyellow dwarf virus (BeYDV). For instance, the rep gene of this virus canbe rendered non-functional, e.g., minimally by inducing a frame shift,in a DNA construct of the present invention. As used herein,“non-functional” means that the protein encoded and expressed by theaffected nucleotide sequence is unable to perform its customary andusual function, in this case rescue and replication of a recognizedsequence. As used herein, “functional” means capable of performing itscustomary and usual function. Inactivation of the native rep gene orremoval of the native rep gene is desired in order to permit inducibleexpression of a separately provided viral replicase gene.

[0179] The invention provides for a pair of DNA constructs wherein afirst DNA construct comprises at least a portion of an LIR of ageminivirus genome, including a site for insertion of a nucleotidesequence encoding a protein of interest, and lacks both a functionalgeminiviral coat protein encoding sequence and a functional rep gene.The rep gene can either be deleted (as shown in FIG. 3C) or inactivated(as shown in FIG. 3A). The second DNA construct comprises a functionalgeminiviral replicase gene encoding a Rep protein, operably linked to afruit ripening-dependent promoter.

[0180] Vectors comprising at least a portion of a geminiviral LIR,including a site for insertion of a nucleotide sequence encoding aprotein of interest, and lacking both a functional geminiviral coatprotein encoding sequence and a functional rep gene useful according tothe invention include pBY024 (FIG. 5).

[0181] A DNA construct comprising a gene of interest flanked by two LIRsuseful according to the invention is pBY217 (FIG. 6), a derivative ofpBY024 wherein the expression cassette for hepatitis B surface antigenis flanked by BeYDV LIR elements.

[0182] Another DNA construct comprising a gene of interest flanked bytwo LIRs useful according to the invention is pBY027 (FIG. 7), aderivative of pBY024 wherein the expression cassette for GFP is flankedby BeYDV LIR elements. Other DNA constructs comprising a gene ofinterest flanked by two LIRs useful according to the invention arepBY028 (FIG. 8), and pBY029 (FIG. 9) a derivative of pBY024 wherein theexpression cassette for GUS (UidA) is flanked by BeYDV LIR elements.pBY028 and pBY029 comprise the expression cassette for GUS (UidA) clonedin a orientation such that the 5′ end of the GUS gene is adjacent to theLIR and such that the 3′ end of the GUS gene is adjacent to the LIR,respectively.

[0183] The invention also provides for a DNA construct comprising atleast a portion of a geminiviral LIR, a restriction site for insertionof a gene of interest, and a functional geminiviral replicase geneoperably linked to a fruit ripening-dependent promoter.

[0184] A vector comprising a DNA construct comprising at least a portionof a geminiviral LIR, a restriction site for insertion of a gene ofinterest, and a functional geminiviral replicase gene operably linked toa fruit ripening-dependent promoter is prepared by cloning a Rep proteinencoding sequence under the control of a fruit ripening-dependentpromoter into pBY024 by methods well known in the art (Ausubel et al.,1995, Short Protocols in Molecular Biology, 3rd Edition, John Wiley &Sons, Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory).

[0185] Since replication of a nucleotide sequence of interest isultimately desired it is necessary to provide a viral replicase,preferably as needed. This can be achieved by supplying a wild-typegeminivirus, or more preferably, by using genetic engineering techniquesto provide a geminiviral rep gene as an expression cassette or a viralreplicon, which rep gene is under the control of a fruitripening-dependent promoter. rep genes useful according to the inventionare derived from any geminivirus included in the section entitled“Geminiviruses”. RepA refers to the 250 amino acid translation productof the C1 open reading frame (ORF). Rep refers to the 360 amino acidtranslation product of the spliced C1 and C2 ORFs. Upon transcriptionand translation, the expressed protein replicase can act in trans toeffect rescue and replication of the desired heterologous nucleotidesequence. A preferred rep gene sequence useful according to theinvention is presented in FIG. 10. Useful Rep constructs include but arenot limited to pBY002 (FIG. 5), p35S-REP (FIG. 11) p2S-REP (FIG. 12),pES-REP (FIG. 13), pBY034 (FIG. 14) and pBY036 (FIG. 15). Useful Repconstructs wherein the rep gene is under the control of the alchoholinducible AlcR promoter include pK!S-ARP(F) (FIG. 16) and pSRN-ARP(F)(FIG. 17). Removal of the intron within the Mastrevirus rep gene greatlyenhances BeYDV replication. A prefered intronless rep gene sequenceuseful according to the invention is presented in FIG. 18. DNAconstructs comprising an intronless form of Mastrevirus Rep (A intron)are also useful according to the invention. Intronless Rep constructsuseful according to the invention include but are not limited top35SΔintron (FIG. 19), p2SΔintron (FIG. 20), pESΔintron (FIG. 21) andpBY035 (FIG. 22). A useful intronless Rep construct comprising anintronless rep gene under the control of the AlcA promoter ispSRN-ADP(F) (FIG. 23). Alternatively, the invention provides for aproviding a geminiviral rep protein under the control of a fruitripening-dependent promoter in cis.

[0186] A DNA construct according to the invention preferably has aplant-functional promoter operably linked to the 5′ end of a nucleotidesequence of interest. A preferred promoter is selected from among CaMV35S, tomato E8, patatin, ubiquitin, mannopine synthase (mas), rice actin1, soybean seed protein glycinin (Gy1), soybean vegetative storageprotein (vsp), and granule-bound starch synthase (gbss). A construct ofthe present invention can also include a translational enhancer region,such as tobacco etch virus (TEV) enhancer, which has been describedelsewhere (Carrington, et al. (1990)). Optionally, a construct of theinvention can comprise at least one vegetative storage protein (VSP)signal peptide encoding sequence, such as an αS or αL sequence (Mason etal. 1988), operably linked to the 5′ end of a nucleotide sequenceencoding a protein of interest.

[0187] As used herein, the term “operably linked” refers to therespective coding sequence being fused in-frame to a promoter, enhancer,termination sequence, and the like, so that the coding sequence isfaithfully transcribed, spliced, and translated, and the otherstructural features are able to perform their respective functions.

[0188] A DNA construct useful according to the invention includes ageminiviral replicase gene encoding a Rep protein under the control of afruit ripening-dependent promoter. A preferred fruit ripening-dependentpromoter is selected from the fruit specific promoter tomato E8(described in Deikman et al., 1992, Plant Physiol., 100:2013), the seedspecific promoter Arabidopsis thaliana 2S-2 (described in Guerche etal., 1990, The Plant Cell, 2:469), or the ethanol-inducible promoter(described in Caddick et al., Nature Biotech., 16:177). Additional fruitripening promoters useful according to the invention include the CaMv35S promoter, the soybean seed protein glycinin (Gy1) promoter, thepatatin tuber specific promoter and the inducible AlcA promoter. Themas, soybean vegetative storage protein (vsp), gbss, glucocorticoid,estrogen, jasmonic acid, insecticide RH5992, copper, tetracycline, andalchohol-inducible promoters are also useful fruit ripening-dependentpromoters according to the invention.

[0189] A nucleotide sequence of interest of the invention is preferablyoperably linked at its 3′ end to a plant-functional terminationsequence. Preferred termination sequences include nopaline synthase(nos), vegetative storage protein (vsp), protease inhibitor 2 (pin2),and geminiviral short intergenic (sir) termination sequences.

[0190] A DNA construct of the invention can be single-stranded, as inthe native geminiviral genome. Also, the DNA can be in itsdouble-stranded replicative form, which includes a complementary strand.As used herein, the term “transgene” refers to a nucleotide sequenceencoding a protein of interest together with the regulatory featuresnecessary to effect transcription of the coding sequence. Such atransgene can be synthesized directly or derived from a genomic or cDNAlibrary, and additionally may be amplified, such as by the polymerasechain reaction (PCR), according to methods well known in the art anddescribed in Maniatis, supra, Ausubel, supra).

[0191] Another aspect of the present invention is an expression vectorcomprising an aforementioned DNA construct of the invention. Such avector includes a selectable marker gene and a multiple cloning siteinto which is inserted a nucleic acid sequence comprising at least aportion of a long intergenic region (LIR) of a geminivirus genomeoperably linked to a nucleotide sequence encoding a protein of interest.Preferably, the nucleic acid sequence lacks a functional geminiviralcoat protein encoding sequence, as produced by deleting all or part ofthis sequence. An expression vector of the invention also preferably hasan E. coli origin of replication, in order to permit the use ofconventional techniques in producing clones of the construct.

[0192] Marker genes useful according to the invention may include a geneencoding a selectable marker, e.g., an antibiotic resistance gene suchas the bacterial tetracycline resistance gene. Incorporation of thetetracycline resistance gene permits the use of tetracycline as aselective agent in the plasmid preparation procedure according to theinvention. One advantage to the use of a tetracycline resistance gene isthat tetracycline is not degraded in E. coli, and therefore moretetracycline does not have to be added during fermentation. In addition,the tetracycline resistance gene is preferred over a gene encodingampicillin resistance because tetracycline is prescribed less often asan antibiotic in a clinical setting, and therefore read through from theplasmid resistance gene will be less likely to interfere with the use ofan antibiotic in a clinical setting.

[0193] Additional marker genes useful according to the invention includeresistance to biocide, particularly an antibiotic, such as kanamycin,G418, bleomycin, hygromycin, chloramphenicol or the like. The particularmarker employed will be one which allows for selection of transformedcells as compared to cells lacking the nucleic acid which has beenintroduced.

[0194] A vector can also have an A. tumefaciens origin of replication,such as when it is desired to maintain the vector in A. tumefaciens forlater transformation with this system. In this event, the nucleotidesequence encoding a protein of interest is flanked by the left and rightT-DNA border regions to effect its transfer to a host plant cell.

[0195] As used herein, the term “vector”, and the like, refers to anucleic acid construct capable of self-replication. Such a vectorincludes a plasmid, bacteria transformed with plasmids, phage vectors,cosmids, and bacterial and yeast artificial chromosomes. Generally, avector of the present invention will be a plasmid, whether it is presentin vitro, in E. coli, in A. tumefaciens, or as a nuclear episome of aplant. Suitable techniques for assembling the instant structuralcomponents into an expression cassette or replicon are described byManiatis et al. (1982).

[0196] Expression vectors useful according to the invention includepBY023 (FIG. 24) and pBY032 (FIG. 25). An expression vector comprisingan expression cassette for hepatitis B surface antigen useful accordingto the invention is pBY217 (FIG. 6).

[0197] A strain of bacteria, such as E. coli, can be transfected with anexpression vector of the present invention in order to grow/amplify aninstant expression cassette according to methods well known in the art(Ausubel, supra, Maniatis, supra). The E. coli can also be mated with A.tumefaciens to introduce the vector therein, where it can reside intactas a shuttle vector. A helper Ti plasmid in the A. tumefaciens canprovide the vir genes necessary to transfer the T-DNA directly from theshuttle vector to the plant cell. Alternatively, the vector can undergohomologous recombination with a tumor-inducing (Ti) plasmid and exchangethe instant cassette for the T-DNA of the Ti plasmid. The inventiontherefore provides for producing transiently transformed plant cellswherein DNA constructs are maintained as episomes. Alternatively, theinvention provides for methods of stably transforming plant cellswherein a DNA construct that is introduced into a plant cell is stablyintegrated into a chromosome.

[0198] Another strain of A. tumefaciens contains an expression vector ofthe present invention and a Ti plasmid that comprises a viral replicaseencoding sequence, e.g., a mastrevirus rep sequence, in its T-DNAsegment. Such a Ti plasmid preferably has the 5′ end of the replicaseencoding sequence operably linked to a fruit ripening-dependentpromoter, which permits inducible replication and amplification of agene of interest in planta. A preferred fruit ripening dependentpromoter useful according to the invention includes but is not limitedto tomato E8, patatin, mas, soybean seed protein glycinin (Gy1), soybeanvegetative storage protein (vsp), gbss, estrogen, jasmonic acid,insecticide RH5992, copper, tetracycline, and alcohol-induciblepromoters. Also, the 3′ end of the replicase encoding sequence ispreferably operably linked to a plant-functional termination sequence,such as a nos, vsp, pin2, or sir termination sequence.

[0199] III. Genes of Interest and Proteins of Interest According to theInvention

[0200] A. Proteins Useful According to the Invention

[0201] Preferred proteins of interest for use with the present inventioninclude reporter molecules, such as firefly luciferase (Genbank #M15077), glucuronidase (GUS) (Genbank #AAC74698), green fluorescentprotein (GFP) (GenBank #E17099), and enhanced versions thereof,particularly for use in optimizing the parameters of this expressionsystem. Proteins of interest useful according to the invention alsoinclude antigenic proteins such as shigatoxin B (StxB) (Genbank#AJ132761), staphylococcus enterotoxin B (SEB) (GenBank #Ml 1118), E.coli labile toxin B (LT-B) (GenBank#AB011677), E. coli labile toxin Asubunit (LT-A) (GenBank #AB011677), Norwalk virus capsid protein (NVCP)(GenBank #AF093797), and hepatitis B surface antigen (HBsAg) (GenBank#AF090842). It is preferred that such antigenic proteins associate asantigenic particles and/or complexes when such association is necessaryto impart immunogenicity thereto. Repeats of aforementioned sequences,e.g., IR sequences, can be employed, when desired, to generate and/orstabilize larger nucleotide sequences and fusion proteins. Proteinsuseful according to the invention also include dimeric IgA, epithelialtransport molecules, monoclonal antibodies and blood substituteproteins.

[0202] Proteins useful according to the methods of the invention alsoinclude but are not limited to proteins that are useful according to theinvention, such as receptors, enzymes, ligands, regulatory factors, andstructural proteins. Therapeutic proteins including nuclear proteins,cytoplasmic proteins, mitochondrial proteins, secreted proteins,plasmalemma-associated proteins, serum proteins, viral antigens andproteins, bacterial antigens, protozoal antigens and parasitic antigensare also useful according to the invention.

[0203] Therapeutic proteins useful according to the invention alsoinclude lipoproteins, glycoproteins, phosphoproteins. Proteins orpolypeptides which can be expressed using the methods of the presentinvention include hormones, growth factors, neurotransmitters, enzymes,clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumorantigens, tumor suppressors, structural proteins, viral antigens,parasitic antigens and bacterial antigens. Specific examples of thesecompounds include proinsulin (GenBank #E00011), growth hormone,dystrophin (GenBank # NM_(—)007124), androgen receptors, insulin-likegrowth factor I (GenBank #NM_(—)00875), insulin-like growth factor II(GenBank #X07868) insulin-like growth factor binding proteins, epidermalgrowth factor TGF-α (GenBank #E02925), TGF-β (GenBank #AW008981), PDGF(GenBank #NM_(—)002607), angiogenesis factors (acidic fibroblast growthfactor (GenBank #E03043), basic fibroblast growth factor (GenBank#NM_(—)002006) and angiogenin (GenBank #M11567), matrix proteins (TypeIV collagen (GenBank #NM_(—)000495), Type VII collagen (GenBank#NM_(—)000094), laminin (GenBank # J03202), phenylalanine hydroxylase(GenBank #K03020), tyrosine hydroxylase (GenBank #X05290), oncogenes(ras (GenBank #AF 22080), fos (GenBank #k00650), myc (GenBank #J00120),erb (GenBank #X03363), src (GenBank #AH002989), sis GenBank #M84453),jun (GenBank #J04111)), E6 or E7 transforming sequence, p53 protein(GenBank #AH007667), Rb gene product (GenBank #m19701), cytokinereceptor, Il-1 (GenBank #m54933), IL-6 (GenBank #e04823), IL-8 (GenBank#119591), viral capsid protein, and proteins from viral, bacterial andparasitic organisms which can be used to induce an immunologic response,and other proteins of useful significance in the body. The compoundswhich can be incorporated are only limited by the availability of thenucleic acid sequence for the protein or polypeptide to be incorporated.One skilled in the art will readily recognize that as more proteins andpolypeptides become identified they can be integrated into the DNAconstructs of the invention and used to transform plant seeds and plantcells and produce transgenic plants, useful for amplification of a geneof interest and overproduction of a protein of interest, according tothe methods of the present invention.

[0204] B. Nucleotide Sequences Useful According to the Invention

[0205] 1. Genes Encoding Toxins

[0206] Examples of genes useful in the invention include those encodingsuch agents including but not limited to genes encoding diphtheriatoxin, Pseudomonas exotoxin, cholera toxin, pertussis toxin, etc., asfollows. Diphtheria toxin-IL2 fusions for inhibition of HIV-1 infection(Zhang et al., 192, Jour. Acquired Immune Deficiency Syndrome 5:1181);Diphtheria toxin A chain for inhibition of HIV viral production(Harrison et al., 1992, AIDS Res. Hum. Retro. 8:39 and Curel et al.,1993, Hum. Gene Ther. 4:71); Diphtheria toxin A chain-liposome complexesfor suppression of bovine leukemia virus infection (Kakidani et al.,1993, Microbiol. Immunol. 37:713); Diphtheria Toxin A chain gene coupledwith immunoglobulin enhancers and promoters for B-cell toxicity (Maxwellet al., Cancer Res., 1991, 51:4299); Tat- and Rev- activated expressionof a diphtheria toxin A gene (Harrison, 1991, Hum. Gene Ther. 2:53);Diphtheria toxin-CD4 fusion for killing of HIV-infected cells (Auilo etal., 1992, Eur. Mol. Biol. Org. Jour. 11:575).

[0207] Other toxins which are useful according to the invention includebut are not limited to the following. Conditionally toxic retrovirusesare disclosed in Brady et al., 1994, Proc. Nat. Aca. Sci. 91:365 and inCaruso et al., 1992, Bone Marrow Transplant, 9:187. Toxins against EBVinfection are disclosed in Harris et al., 1991, Cell. Immunol. 134:85,and against poliovirus in Rodriguez et al., 1992, Jour. Virol. 66:1971.Toxins against influenza virus are disclosed in Bron et al., 1994,Biochemistry 33:9110.

[0208] 2. Genes Encoding Immunoactive Agents

[0209] Another agent useful according to the invention includesimmunoactive agents, i.e., agents which combat viral infections orproduction by activating an immune response to the virus. Such agentsinclude but are not limited to cytokines against viruses in general(Biron, 1994, Curr. Opin. Immunol. 6:530); soluble CD4 against SIV(Watanabe et al., 1991, Proc. Nat. Aca. Sci. 88:126); CD4-immunoglobulinfusions against HIV-1 and SIV (Langner et al., 1993, Arch. Virol.130:157); CD4(81-92)-based peptide derivatives against HIV infection(Rausch et al., 1992, Biochem. Pharmacol. 43:1785); lympho-cytotoxicantibodies against HIV infection (Szabo et al., 1992, Acta. Virol.38:392); IL-2 against HIV infection (Bell et al., 1997, Clin Exp.Immunol. 90:6); and anti-T cell receptor antibodies against viruses ingeneral (Newell et al., 1991, Ann. N.Y. Aca. Sci. 636:279).

[0210] 3. Genes Encoding Anti-Viral Drugs

[0211] Genes encoding anti-viral agent useful according to the inventioninclude genes encoding drugs having anti-viral activity and which arethe direct product of a gene or are a product of a gene encoding aprecursor of the drug, the drug then being synthesized by a biosyntheticpathway in the cell. Targets of drug intervention in the replicativecycle of, for example, a retrovirus, include (1) binding and entry, (2)reverse transcriptase, (3) transcription and translation, and (4) viralmaturation and budding. Representative inhibitors of viral binding andentry for HIV include recombinant soluble CD4, immunoadhesions, peptideT, and hypericin. Nucleoside reverse transcriptase inhibitors includezidovudine, didanosine, zalcitabine, and starudine. Foscarnet,tetrahydroimidazobenzodiazepinethione compounds, and nevirapine are somenon-nucleoside reverse transcriptase inhibitors. Inhibitors oftranscription and translation include antagonists of the TAT gene andGLQ223. Castanospernine and protease inhibitors interfere with viralbudding and maturation. Such drugs include but are not limited tonucleoside or nucleotide analogs and products of a cellular biosyntheticpathway such as described in Harrell et al., 1994, Drug Metab. Dispos.22:124 (deoxy-guanine); Fillon et al., 1993, Clin. Invest. Med. 16:339(dauno-rubicin); Ohrvi et al., 1990, Nucleic Acids Symp. 26:93(anti-viral nucleosides); Hudson et al., 1993, Photochem. Photobiol.57:675 (thiarubines); Salhany et al., 1993, Jour. Biol. Chem. 268:7643(pyridoxal 5′-phosphate); Damaso et al., 1994, Arch. Viral. 134:303(cyclosporin A); Gallicchio et al., 1993. Int. Jour. Immunol. 15:263(dideoxynucleoside drugs); and Fiore et al., 1990, Biol. Soc. Ital.Biol. Sper. 66:601 (AZT).

[0212] For many of these and other non-plant proteins, the nucleotidesequence encoding the protein is preferably optimized for expression inplants, e.g., by introducing one or more codons degenerate to thecorresponding native codon. Other plant-optimization measures for codingsequences include removal of spurious mRNA processing signals such aspolyadenylation signals, splicing sites, and transcription terminationsignals, removal of mRNA destabilizing sequences, removal of thecytosine methylation motif “CCGG”, modifying the translation start siteand introducing a C-terminal KDEL signal, such as SEKDEL(Ser-Glu-Lys-Asp-Glu-Leu), which presumably aids return of the nascentprotein to the endoplasmic reticulum for processing.

[0213] A nucleotide sequence of interest of the present invention can beprovided as its wild-type sequence. Alternatively, a synthetic sequence,such as a “plant-optimized” sequence mentioned above can be employed. Anucleotide sequence having a high degree of homology to these sequences,so that the encoded amino acid sequence remains substantially unchanged,are also contemplated. In particular, sequences at least 80%, morepreferably 90%, homologous with an aforementioned nucleotide sequenceare contemplated. It should be noted, however, that only that thoseepitopes of an expressed antigenic protein essential for generating thedesired immune response need be present in the translated molecule.Accordingly, C- and/or N-terminal fragments, including portions offusion proteins, presenting the essential epitopes are contemplatedwithin the invention. Such fragments can be encoded in a vectorconstruct of the invention or can be generated in vivo or in vitro bypost-translation cleavage processes.

[0214] Additional proteins of interest useful according to the inventioninclude Hepatitis B Surface Antigen, Norwalk Capsid Protein and E. coliheat-labile enterotoxin.

[0215] Hepatitis B Surface Antigen (HBsAg)

[0216] The feasibility of expressing HBsAg as virus-like particles(VLPs) in tobacco leaves has been demonstrated (Mason et al., 1992). TheVLPs are similar to the recombinant yeast-derived vaccine, which islicensed for parenteral immunization. The formation of HBsAg particlesrequires insertion of the peptide in the endoplasmic reticulum (ER)membrane with four transmembrane domains, followed by budding ofparticles into the ER lumen. The plant-derived HBsAg retains both B- andT-cell epitopes when studied in a mouse model (Thanavala et al., 1995).The finding that plant cells can produce an immunogenic HBsAg VLPindicates that plants are a feasible expression system for animalproteins that assemble into complex structures. Accordingly, thenecessary genetic elements for generating immunogenic HBsAg particleshave been identified. These elements, e.g., regulatory and codingregions, can be incorporated into a geminiviral vector as describedherein to afford a means of amplifying expression of this antigen inplants.

[0217] Norwalk Capsid Protein (NVCP)

[0218] The expression and VLP assembly in plants of NVCP, and its oralimmunogenicity in mice have been reported (Mason et al., 1996). The NVCPaccumulated to 0.3% of the total protein, and assembled into VLPs withabout 60% efficiency in tobacco leaf and potato tuber cells. When viewedby negative staining electron microscopy, the empty capsids werevirtually indistinguishable from those produced in an insect cellsystem. Further, the material was orally immunogenic in mice when givenby gavage in 4 doses as low as 10 μg each, or when given by directfeeding of potato tuber slices in 4 doses as low as 50 μg each. Bothserum IgG and gut mucosal IgA were stimulated by the plant vaccine.Thus, the means for generating immunogenic amounts of VLPs of NVCPantigens have been identified. These can be used with the presentinvention to generate yet higher levels of immunogen in plants accordingto the gene amplification methods described herein.

[0219]E. coli Heat-Labile Enterotoxin (LT)

[0220] LT is a potent mucosal immunogen and adjuvant that stimulatesimmune responses against co-administered antigens (Clements et al.,1988). The B-subunit of LT (LT-B) expressed in tobacco leaves assemblesinto active oligomers that possess ganglioside G_(M1) binding capacity(Haq et al., 1995). It is found that addition of a microsomal retentionsequence (SEKDEL) at the carboxyl-terminus of LT-B increases itsaccumulation in plant tissue, while still allowing G_(M1) binding andimmunogenicity. In oral tests with plant-derived LT-B given to mice,either tobacco leaf extracts administered by gavage or potato tubers fedwithout preparation (other than slicing) stimulated serum and gutmucosal antibodies against LT-B (Haq et al., 1995). The serum anti-LT-Bfrom these animals showed inhibition of LT activity, indicating itspotential value as a protective vaccine. In further experiments, greatenhancement of LT-B expression is obtained when a plant codon-optimizedgene is used (Table 1) (Mason et al. 1998). The necessary geneticmachinery for expressing single copies of the LT-B gene in plants hastherefore been identified. This approach can now be used in conjunctionwith the methods disclosed herein to achieve still higher levels ofexpression by way of gene amplification. TABLE 1 Expression of differentLT-B coding regions in potato leaves Soluble LT-B, Coding region(plasmid) Modification ng/mg Native LT-B (pLTB110) Optimize translation 70 start site LTB-SEKDEL (pLTK110) C-terminal SEKDEL  190 extensionSynthetic LT-B (pTH110) Plant-optimize codons, 4640 eliminate spuriouspolyadenylation and splicing signals

[0221] Oral Adjuvants

[0222] An edible vaccine of the present invention entails providing atleast a portion of a transgenic plant generated as described herein.Preferably, the vaccine further comprises an immunologically acceptableadjuvant, i.e., one that promotes an immune response to the antigenicprotein expressed by the plant without producing a serious deleteriouseffect. Preferred adjuvants include cholera toxin (CT), heat-labileenterotoxin (LT), anti-idiotypic antibody 2F 10, colonization factor,shiga-like toxin, intimin, and mutants thereof.

[0223] The expression of E. coli heat-labile enterotoxin (LT) in plantsas an oral adjuvant when co-expressed with other vaccine antigens hasbeen explored. The goal is to produce assembled and active holotoxin LT(A₁B₅) in edible tissues. In early experiments using the nativebacterial LT-A and LT-B coding sequences, potato transformants thatexpress mRNAs (detected by Northern blot of total tuber RNA) for bothLT-A and LT-B were detected, but holotoxin was not detected.

[0224] IV. Methods of Introducing Nucleic Acid into Cells

[0225] A. Transformation methods

[0226] Methods of gene transfer into plants include use of the A.tumefaciens—Ti plasmid system. The tumor-inducing (Ti) plasmids of A.tumefaciens contain a segment of plasmid DNA called transforming DNA(T-DNA), which integrates into the plant host genome. First, a plasmidvector is constructed that replicates in E. coli. This plasmid containsthe DNA encoding the protein of interest and this DNA is flanked byT-DNA border sequences, which define the points at which the DNAintegrates into the plant genome. Usually a gene encoding a selectablemarker (such as a gene encoding resistance to an antibiotic such askanamycin) is also inserted between the left border (LB) and rightborder (RB) sequences. The expression of this gene in transformed plantcells gives a positive selection method to identify those plants orplant cells having an integrated T-DNA region. Second, the plasmid istransferred to Agrobacterium. This can be accomplished via a conjugationmating system, or by direct uptake of plasmid DNA by the Agrobacterium.For subsequent transfer of the T-DNA to plants, the Agrobacterium strainutilized must contain a set of inducible virulence

[0227] (vir) genes, which are essential for T-DNA transfer to plantcells.

[0228] The A. tumefaciens gene transfer system mentioned above is theetiologic agent of crown gall, a disease of a wide range of dicotyledonsand gymnosperms [DeCleene, M. et. al., Bot. Rev. 42, 389 (1976)], thatresults in the formation of tumors or galls in plant tissue at the sitethe infection. The Agrobacterium system has been developed to permitroutine transformation of a variety of plant tissue [see, e.g., Schell,J. et al., Bio/Technology 1, 175 (1983); Chilton, M-D, ScientificAmerican 248, 50 (1983)]. Representative tissues transformed in thismanner include tobacco [Barton, K. et al., Cell 32, 1033 (1983)]; tomato[Fillatti, S. et al., Bio/Technology 5, 726 (1987)]; sunflower [Everett,N. et al., Bio/Technology 5, 1201 (1987)]; cotton [Umbeck, P. et al.,Bio/Technology 5, 263 (1987)]; rapeseed [Pua, E. et al., Bio/Technology5, 815 (1987)]; potato [Facciotti D. et al., Bio/Technology 3, 241(1985); poplar [Pythoud, F. et al., Bio/Technology 5, 1323 (1987); andsoybean [Hinchee, M. et al., Bio/Technology 6, 915 (1988)]. Other plantscan be transformed by routine extensions or modifications of thesemethods.

[0229] Multiple choices of Agrobacterium strains and plasmidconstruction strategies can be used to optimize genetic transformationof plants. For instance, A. tumefaciens may not be the onlyAgrobacterium strain used. Other Agrobacterium strains such as A.rhizogenes may be more suitable in some applications. A. rhizogenes,which incites root hair formation in many dicotyledonous plant species,carries a large extra-chromosomal element called an Ri (root-including)plasmid, which functions in a manner analogous to the Ti plasmid of A.tumefaciens. Transformation using A. rhizogenes has developedanalogously to that of A. tumefaciens and has been successfully utilizedto transform, for example, alfalfa, [Sukhapinda, K. et al., Plant Mol.Biol. 8, 209 (1987)].

[0230] Methods of inoculation of the plant tissue vary depending uponthe plant species and the Agrobacterium delivery system. A convenientapproach is the leaf disc procedure which can be performed with anytissue explant that provides a good source for initiation of whole plantdifferentiation. The addition of nurse tissue may be desirable undercertain conditions. Other procedures such as in vitro transformation ofregenerating protoplasts with A. tumefaciens may be followed to obtaintransformed plant cells as well.

[0231] Several so-called “direct” gene transfer procedures have beendeveloped to transform plants and plant tissues without the use of anAgrobacterium intermediate. Plant regeneration from protoplasts is aparticularly useful technique [Evans, D. A. et al., Handbook of PlantCell Culture 1, 124 (1983)]. When a plant species can be regeneratedfrom protoplasts, direct gene transfer procedures can be utilized andtransformation is not dependent on the use of A. tumefaciens. In thedirect transformation of protoplasts the uptake of exogenous geneticmaterial into a protoplast may be enhanced by use of a chemical agent orelectric field. The exogenous material may then be integrated into thenuclear genome.

[0232] Early work has been conducted in the dicot Nicotiana tabacum(tobacco) where it was shown that the foreign DNA was incorporated andtransmitted to progeny plants [Paszkowski, J. et al., EMBO J, 3: 2717(1984); Potrykus, I. et al., Mol. Gen. Genet. 199: 169 (1985)]. Monocotprotoplasts have also been transformed by this procedure: for example,Triticum monococum [Lorz H. et al., Mol. Gen. Genet. 199: 178 (1985)];Lolium multiflorum (Italian ryegrass), Potrykus, I. et. al., Mol. Gen.Genet 199, 183 (1985); maize [Rhodes, C., et al., Bio/Technology 5, 56(1988)]; and Black Mexican sweet corn [Fromm, M. et al., Nature 319, 719(1986)]. Other plants that have been regenerated from protoplastsinclude rice [Abdulah, R. et al., Bio/Technology 4, 1987 (1987)];rapeseed [Kansha, et al., Plant Cell Reports 5, 101 (1986)]; potato[Tavazza, R. et al., Plant Cell Reports 5, 243 (1986)]; eggplant,Sihachaki, D. et al., Plant Cell, Tissue, Organ Culture 11, 179 (1987);and cucumber [Jia, S-R., et al., J. Plant Physiol. 124, 393 (1986)].Methods for directly transforming protoplasts of other varieties areevident.

[0233] Introduction of DNA into protoplasts of a plant can be effectedby treatment of the protoplasts with an electric pulse in the presenceof the appropriate DNA in a process called electroporation. In thismethod, the protoplasts are isolated and suspended in a mannitolsolution. Supercoiled or circular plasmid DNA is added. The solution ismixed and subjected to a pulse of about 400 V/cm at room temperature forless than 10 to 100 microseconds. A reversible physical breakdown of themembrane occurs to permit DNA uptake into the protoplasts.

[0234] DNA viruses have been used as gene vectors in plants. Acauliflower mosaic virus carrying a modified bacterialmethotrexate-resistance gene was used to infect a plant. The foreigngene was systematically spread in the plant [Brisson, N. et al., Nature310, 511 (1984)]. The advantages of this system are the ease ofinfection, systematic spread within the plant, and multiple copies ofthe gene per cell.

[0235] Liposome fusion has also been shown to be a method fortransformation of plant cells. In this method, protoplasts are broughttogether with liposomes carrying the desired gene. As membranes merge,the foreign gene is transferred to the protoplasts [Dehayes, A. et al.,EMBO J. 4, 2731 (1985)].

[0236] Polyethylene glycol (PEG) mediated transformation has beencarried out in N. tabacum (a dicot) and Lolium multiflorum (a monocot).It is a chemical procedure of direct gene transfer based on synergisticinteraction between Mg²⁺, PEG, and possibly Ca²⁺ [Negrutiu, R. et al.,Plant Mol. Biol. 8, 363 (1987)]. Alternatively, exogenous DNA can beintroduced into cells or protoplasts by microinjection. A solution ofplasmid DNA is injected directly into the cell with a finely pulledglass needle.

[0237] A recently developed procedure for direct gene transfer involvesbombardment of cells by microprojectiles carrying DNA [Klein, T. M. etal., Nature 327, 70 (1987)]. In this “biolistic” procedure, tungsten orgold particles coated with the exogenous DNA are accelerated toward thetarget cells. At least transient expression has been achieved in onion.This procedure has been utilized to introduce DNA into Black Mexicansweet corn cells in suspension culture and maize immature embryos andalso into soybean protoplasts [Klein, T. M. et al., Bio/Technology 6,559 (1988)]. Stably transformed cultures of maize and tobacco have beenobtained by microprojectile bombardment. Stably transformed soybeanplants have been obtained by this procedure [McCabe, D. E. et al.,Bio/Technology 6, 923 (1988)].

[0238] To produce transformed seeds, flowers of Arabidopsis aretransformed according to the following method. The Agrobacterium isvacuum-infiltrated into developing flowers, and the resulting seed arethen screened for marker resistance and foreign gene expression.Presumably, stamens/pollen, ovary/egg, or even the developing zygote iffertilization has already occurred are transformed. This method(described in Clough & Bent, 1998, Plant J., 16:735) is used totransform Arabidopsis with a construct comprising a rep gene under thetranscriptional regulation of the At2S-2 seed promoter.

[0239] V. Plants, Cells and Seeds Useful According to the Invention

[0240] Plants that can be used for practice of the present inventioninclude any dicotyledon and monocotyledon. These include, but are notlimited to, tobacco, carrot, spinach, pepper, potato, tomato, apple,wheat, rye, soybean, rice, maize, corn, berries such as strawberries,raspberries, alfalfa and banana. Since many edible plants used by humansfor food or as components of animal feed are dicotyledenous plants,dicotyledons are typically employed, although monocotyledontransformation is also applicable especially in the production ofcertain grains useful for animal feed. It is particularly advantageousin certain disease prevention for human infants to produce a vaccine ina juice for ease of administration to humans such as juice of tomato,soybean, and carrot, or milk. Cells and seeds derived from these plantvaccines are also useful according to the invention.

[0241] A transgenic plant transformed with a vector describedhereinabove is another aspect of the present invention. Particularlypreferred plant hosts for the vector include banana, tomato, potato andcarrot.

[0242] Potato varieties FL 1607 (“Frito Lay 1607”) and Desiree, andtomato variety Tanksley TA234TM2R are particularly preferred varieties,which have been transformed with binary vectors using the methodsdescribed herein. Of these transformed varieties, Desiree is the onlycommercial variety; the other varieties can be obtained from Frito-Lay(Rhinelander, Wis.) and Steve Tanksley (Dept. of Plant Breeding, CornellUniv.). Potato variety FL1607 allows rapid transformation but is not agood agronomic variety as it suffers from hollow heart.

[0243] Tomato is preferred as a model system for expression of foreignproteins because of its ease of genetic transformation, and becausefruit-specific, ripening dependent promoters are available for regulatedexpression (Giovannoni et al., 1989). The E8 promoter has been used tomediate high level production of polygalacturonase protein in mutanttomato fruit (Giovannoni et al., 1989) and monellin in wild-type tomatofruit (Penarrubia et al., 1992). Tomato is a host for BeYDV (Palmer &Rybicki, 1997) and will therefore support Rep-mediated replication ofviral DNA.

[0244] The important points for virus-mediated gene amplification intomato, before developing the system in a more appropriate high-proteinsystem such as soybeans, can be demonstrated. Soybean transformation ismuch more difficult than tomato, so it is prudent to show feasibility inthe more tractable system. Upon showing the salient features of thesystem in soybeans, i.e., Rep protein expression in seeds and rescue ofa vaccine antigen gene in a viral replicon, amplification of thetransgene in soybean is established.

[0245] VI. Methods of Detecting Nucleic Acid and Protein According tothe Invention

[0246] The invention provides for methods of detecting rescue andreplication including but not limited to Southern and northern blotanalysis, PCR-based methods of detection, as well as immunologicalmethods of detecting a protein of interest according to the invention.

[0247] A. Detection of a Nucleotide Sequence of Interest

[0248] 1. Southern Blot Analysis

[0249] Southern blot analysis can be used to detect a nucleotidesequence of interest from a PCR amplified product or from a totalgenomic DNA test sample via a non-PCR based assay. The method ofSouthern blot analysis is well known in the art (Ausubel et al., supra,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual., 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).This technique involves the transfer of DNA fragments from anelectrophoresis gel to a membrane support resulting in theimmobilization of the DNA fragments. The resulting membrane carries asemipermanent reproduction of the banding pattern of the gel.

[0250] Southern blot analysis is performed according to the followingmethod. Genomic DNA (5-20 μg) is digested with the appropriaterestriction enzyme and separated on a 0.6-1.0% agarose gel in TAEbuffer. The DNA is transferred to a commercially available nylon ornitrocellulose membrane (e.g. Hybond-N membrane, Amersham, ArlingtonHeights, Ill.) by methods well known in the art (Ausubel et al., supra,Sambrook et al., supra). Following transfer and UV cross linking, themembrane is hybridized with a radiolabeled probe in hybridizationsolution (e.g. under stringent conditions in 5× SSC, 5× Denhardtsolution, 1% SDS) at 65° C. Alternatively, high stringency hybridizationcan be performed at 68° C. or in a hybridization buffer containing adecreased concentration of salt, for example 0.1× SSC. The hybridizationconditions can be varied as necessary according to the parameters knownin the art. Following hybridization, the membrane is washed at roomtemperature in 2× SSC/0.1% SDS and at 65° C. in 0.2× SSC/0.1% SDS, andexposed to film. The stringency of the wash buffers can also be varieddepending on the amount of the background signal (Ausubel et al.,supra).

[0251] Detection of a nucleic acid probe-target nucleic acid hybrid willinclude the step of hybridizing a nucleic acid probe to the DNA target.This probe may be radioactively labeled or covalently linked to anenzyme such that the covalent linkage does not interfere with thespecificity of the hybridization. A resulting hybrid can be detectedwith a labeled probe. Methods for radioactively labeling a probe includerandom oligonucleotide primed synthesis, nick translation or kinasereactions (see Ausubel et al., supra). Alternatively, a hybrid can bedetected via non-isotopic methods. Non-isotopically labeled probes canbe produced by the addition of biotin or digoxigenin, fluorescentgroups, chemiluminescent groups (e.g. dioxetanes, particularly triggereddioxetanes), enzymes or antibodies. Typically, non-isotopic probes aredetected by fluorescence or enzymatic methods. Detection of aradiolabeled probe-target nucleic acid complex can be accomplished byseparating the complex from free probe and measuring the level ofcomplex by autoradiography or scintillation counting. If the probe iscovalently linked to an enzyme, the enzyme-probe-conjugate-targetnucleic acid complex will be isolated away from the free probe enzymeconjugate and a substrate will be added for enzyme detection. Enzymaticactivity will be observed as a change in color development orluminescent output resulting in a 10³-10⁶ increase in sensitivity. Anexample of the preparation and use of nucleic acid probe-enzymeconjugates as hybridization probes (wherein the enzyme is alkalinephosphatase) is described in (Jablonski et al., 1986, Nuc. Acids Res.,14:6115)

[0252] Two-step label amplification methodologies are known in the art.These assays are based on the principle that a small ligand (such asdigoxigenin, biotin, or the like) is attached to a nucleic acid probecapable of specifically binding to a gene of interest.

[0253] According to the method of two-step label amplification, thesmall ligand attached to the nucleic acid probe will be specificallyrecognized by an antibody-enzyme conjugate. For example, digoxigeninwill be attached to the nucleic acid probe and hybridization will bedetected by an antibody-alkaline phosphatase conjugate wherein thealkaline phosphatase reacts with a chemiluminescent substrate. Formethods of preparing nucleic acid probe-small ligand conjugates, see(Martin et al., 1990, BioTechniques, 9:762). Alternatively, the smallligand will be recognized by a second ligand-enzyme conjugate that iscapable of specifically complexing to the first ligand. A well knownexample of this manner of small ligand interaction is the biotin avidininteraction. Methods for labeling nucleic acid probes and their use inbiotin-avidin based assays are described in Rigby et al., 1977, J. Mol.Biol., 113:237 and Nguyen et al., 1992, BioTechniques, 13:116).

[0254] Variations of the basic hybrid detection protocol are known inthe art, and include modifications that facilitate separation of thehybrids to be detected from extraneous materials and/or that employ thesignal from the labeled moiety. A number of these modifications arereviewed in, e.g., Matthews & Kricka, 1988, Anal. Biochem., 169:1;Landegren et al., 1988, Science, 242:229; Mittlin, 1989, Clinical Chem.35:1819; U.S. Pat. No. 4,868,105, and in EPO Publication No. 225,807.

[0255] 2. Northern Blot Analysis

[0256] The method of Northern blotting is well known in the art. Thistechnique involves the transfer of RNA from an electrophoresis gel to amembrane support to allow the detection of specific sequences in RNApreparations.

[0257] Northern blot analysis is performed according to the followingmethod. An RNA sample (prepared by the addition of MOPS buffer,formaldehyde and formamide) is separated on an agarose/formaldehyde gelin 1× MOPS buffer. Following staining with ethidium bromide andvisualization under ultra violet light to determine the integrity of theRNA, the RNA is hydrolyzed by treatment with 0.05M NaOH/1.5MNaClfollowed by incubation with 0.5M Tris-Cl (pH 7.4)/1.5M NaCl. The RNA istransferred to a commercially available nylon or nitrocellulose membrane(e.g. Hybond-N membrane, Amersham, Arlington Heights, Ill.) by methodswell known in the art (Ausubel et al., supra, Sambrook et al., supra).Following transfer and UV cross linking, the membrane is hybridized witha radiolabeled probe in hybridization solution (e.g. in 50%formamide/2.5% Denhardt's/100-200 mg denatured salmon sperm DNA/0.1%SDS/5× SSPE) at 42° C. The hybridization conditions can be varied asnecessary as described in Ausubel et al., supra and Sambrook et al.,supra. Following hybridization, the membrane is washed at roomtemperature in 2× SSC/0.1% SDS, at 42° C. in 1× SSC/0.1% SDS, at 65° C.in 0.2× SSC/0.1% SDS, and exposed to film. The stringency of the washbuffers can also be varied depending on the amount of background signal(Ausubel et al., supra).

[0258] 3.PCR

[0259] Nucleic acid sequences of interest of the invention are amplifiedfrom genomic DNA or other natural sources by the polymerase chainreaction (PCR). PCR methods are well-known to those skilled in the art.

[0260] PCR provides a method for rapidly amplifying a particular DNAsequence by using multiple cycles of DNA replication catalyzed by athermostable, DNA-dependent DNA polymerase to amplify the targetsequence of interest. PCR requires the presence of a nucleic acid to beamplified, two single stranded oligonucleotide primers flanking thesequence to be amplified, a DNA polymerase, deoxyribonucleosidetriphosphates, a buffer and salts.

[0261] The method of PCR is well known in the art. PCR, is performed asdescribed in Mullis and Faloona, 1987, Methods Enzymol., 155: 335,herein incorporated by reference.

[0262] PCR is performed using template DNA (at least 1 82 g; moreusefully, 1-1000 ng) and at least 25 pmol of oligonucleotide primers. Atypical reaction mixture includes: 2 μl of DNA, 25 pmol ofoligonucleotide primer, 2.5 μl of 10× PCR buffer 1 (Perkin-Elmer, FosterCity, Calif.), 0.4 μl of 1.25 μM dNTP, 0.15 μl (or 2.5 units) of Taq DNApolymerase (Perkin Elmer, Foster City, Calif.) and deionized water to atotal volume of 25 μl. Mineral oil is overlaid and the PCR is performedusing a programmable thermal cycler.

[0263] The length and temperature of each step of a PCR cycle, as wellas the number of cycles, are adjusted according to the stringencyrequirements in effect. Annealing temperature and timing are determinedboth by the efficiency with which a primer is expected to anneal to atemplate and the degree of mismatch that is to be tolerated. The abilityto optimize the stringency of primer annealing conditions is well withinthe knowledge of one of moderate skill in the art. An annealingtemperature of between 30° C. and 72° C. is used. Initial denaturationof the template molecules normally occurs at between 92° C. and 99° C.for 4 minutes, followed by 20-40 cycles consisting of denaturation(94-99° C. for 15 seconds to 1 minute), annealing (temperaturedetermined as discussed above; 1-2 minutes), and extension (72° C. for 1minute). The final extension step is generally carried out for 4 minutesat 72° C., and may be followed by an indefinite (0-24 hour) step at 4°C.

[0264] Several techniques for detecting PCR products quantitativelywithout electrophoresis may be useful according to the invention. One ofthese techniques, for which there are commercially available kits suchas Taqman™ (Perkin Elmer, Foster City, Calif.), is performed with atranscript-specific antisense probe. This probe is specific for the PCRproduct (e.g. a nucleic acid fragment derived from a gene of interest)and is prepared with a quencher and fluorescent reporter probe complexedto the 5′ end of the oligonucleotide. Different fluorescent markers canbe attached to different reporters, allowing for measurement of twoproducts in one reaction. When Taq DNA polymerase is activated, itcleaves off the fluorescent reporters of the probe bound to the templateby virtue of its 5′-to-3′ nucleolytic activity. In the absence of thequenchers, the reporters now fluoresce. The color change in thereporters is proportional to the amount of each specific product and ismeasured by a fluorometer; therefore, the amount of each color can bemeasured and the PCR product can be quantified. The PCR reactions can beperformed in 96 well plates so that multiple samples can be processedand measured simultaneously. The Taqman™ system has the additionaladvantage of not requiring gel electrophoresis and allows forquantification when used with a standard curve.

[0265] B. Detection of a Protein Sequence of Interest

[0266] 1. Preparation of Antibodies

[0267] Antibodies specific for the proteins of interest of the inventionare useful for protein purification and detection. By antibody, weinclude constructions using the binding (variable) region of such anantibody, and other antibody modifications. Thus, an antibody useful inthe invention may comprise a whole antibody, an antibody fragment, apolyfunctional antibody aggregate, or in general a substance comprisingone or more specific binding sites from an antibody. The antibodyfragment may be a fragment such as an Fv, Fab or F(ab′)₂ fragment or aderivative thereof, such as a single chain Fv fragment. The antibody orantibody fragment may be non-recombinant, recombinant or humanized. Theantibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and soforth. In addition, an aggregate, polymer, derivative and conjugate ofan immunoglobulin or a fragment thereof can be used where appropriate.

[0268] Although a protein product (or fragment or oligopeptide thereof)of a gene of interest of the invention that is useful for the productionof antibodies does not require biological activity, it must beantigenic. Peptides used to induce specific antibodies may have an aminoacid sequence consisting of at least five amino acids and preferably atleast 10 amino acids. Preferably, they should be identical to a regionof the natural protein and may contain the entire amino acid sequence ofa small, naturally occurring molecule. Short stretches of amino acidscorresponding to the protein of interest of the invention may be fusedwith amino acids from another protein such as keyhole limpet hemocyaninor GST, and antibody will be produced against the chimeric molecule.Procedures well known in the art can be used for the production ofantibodies to the proteins of interest of the invention.

[0269] For the production of antibodies, various hosts including goats,rabbits, rats, mice etc . . . may be immunized by injection with theprotein products (or any portion, fragment, or oligonucleotide thereofwhich retains immunogenic properties) of the genes of interest of theinvention. Depending on the host species, various adjuvants may be usedto increase the immunological response. Such adjuvants include but arenot limited to Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parviimare potentially useful human adjuvants.

[0270] a. Polyclonal Antibodies.

[0271] The antigen protein may be conjugated to a conventional carrierin order to increase its immunogenicity, and an antiserum to thepeptide-carrier conjugate will be raised. Coupling of a peptide to acarrier protein and immunizations may be performed as described (Dymeckiet al., 1992, J. Biol. Chem., 267: 4815). The serum can be titeredagainst protein antigen by ELISA (below) or alternatively by dot or spotblotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods, 51: 317).At the same time, the antiserum may be used in tissue sections preparedas described. A useful serum will react strongly with the appropriatepeptides by ELISA, for example, following the procedures of Green etal., 1982, Cell, 28: 477.

[0272] b. Monoclonal Antibodies.

[0273] Techniques for preparing monoclonal antibodies are well known,and monoclonal antibodies may be prepared using a candidate antigenwhose level is to be measured or which is to be either inactivated oraffinity-purified, preferably bound to a carrier, as described byArnheiter et al., 1981, Nature, 294;278.

[0274] Monoclonal antibodies are typically obtained from hybridomatissue cultures or from ascites fluid obtained from animals into whichthe hybridoma tissue was introduced.

[0275] Monoclonal antibody-producing hybridomas (or polyclonal sera) canbe screened for antibody binding to the target protein.

[0276] 2. Antibody Detection Methods

[0277] Particularly preferred immunological tests rely on the use ofeither monoclonal or polyclonal antibodies and include enzyme-linkedimmunoassays (ELISA), immunoblotting and immunoprecipitation (seeVoller, 1978, Diagnostic Horizons, 2:1, Microbiological AssociatesQuarterly Publication, Walkersville, Md.; Voller et al., 1978, J. Clin.Pathol., 31: 507; U.S. Reissue Pat. No. 31,006; UK Patent 2,019,408;Butler, 1981, Methods Enzymol., 73: 482; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.) or radioimmunoassays (RIA)(Weintraub, B., Principles of radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March 1986,pp.1-5, 46-49 and 68-78). For analysing plants for the presence orabsence of a protein of interest according to the present invention,immunohistochemistry techniques may be used. It will be apparent to oneskilled in the art that the antibody molecule may have to be labelled tofacilitate easy detection of a target protein. Techniques for labellingantibody molecules are well known to those skilled in the art (seeHarlow and Lane, 1989, Antibodies, Cold Spring Harbor Laboratory).

[0278] VII. Uses

[0279] A. Gene Amplification

[0280] The constructs of the present invention can be used to amplify agene of interest. According to the method of the invention, a gene ofinterest is amplified in the presence of a Rep protein, preferably about10-fold, more preferably about 30-fold and most preferably about100-fold, as compared to a gene of interest in the absence of a Repprotein.

[0281] B. Protein Production

[0282] The constructs of the present invention can be used tooverproduce a protein of interest. According to the method of theinvention, a protein of interest is overproduced in the presence of aRep protein such that the amount of protein produced is preferably about2%, more preferably about 10% and most preferably about 30% of the totalprotein of a cell comprising a construct of the invention that includesa nucleotide sequence encoding the protein of interest, and wherein thecell further comprises a Rep protein.

[0283] The invention will now be described with reference to certainexamples, which illustrate but do not limit it.

EXAMPLES Example 1 Verification of Rescue and Replication of aChromosome-Integrated Rep-Defective Viral Genome with BeYDV Rep Proteinin Trans

[0284] Transgenic tobacco (Nicotiana benthamian, Nicotiana tabacum)plant lines are created by transformation with wild-type BeYDV partialdimer in a T-DNA binary vector (pBin19) using A. tumefaciens LBA4404.The plasmid pBeYDV1.4/Bin19 is described in Liu et al., 1998, J. Gen.Virol., 79:2265. This plasmid includes the BeYDV 1.4-mer genomecontaining two LIR regions inserted into pBin19 (Bevan, 1984, Nuc.Acids. Res., 12:8711. As expected, the BeYDV genome integrates into thenuclear genomes of the plant cells. Some of the kanamycin-resistanttransgenic plants displayed a stunty phenotype suggesting virusinfection, while other lines appeared normal. DNA was isolated fromplant leaves.

[0285] A Southern blot of plant leaf DNA was probed with labeled BeYDVgenomic DNA, which revealed the presence of replicative forms of BeYDVDNA in the stunty plants. Normal plants, as well as plants transformedwith the pBin19 vector, showed an absence of replicative forms of theDNA. It is believed that in those non-replicating transgenic lines, thesite of insertion into the plant chromosome may limit expression of theRep protein. Electron microscopy of thin sections of the leaves fromstunty transgenic lines revealed the presence of crystalline arrays ofvirions characteristic of geminivirus-infected plants, and negativelystained leaf extracts showed geminate virus particles. These resultsdemonstrate that integrated BeYDV genomic DNA can be rescued andreplicated in some transgenic lines.

Example 2 Plant-Optimized Genes for Shigatoxin B Subunit (StxB) andStaphylococcal enterotoxin B (FB).

[0286] StxB and SEB native sequences were scanned for codon use and forpotential problem sequences, including spurious mRNA processing signalssuch as polyadenylation signals, splice sites, transcription terminationsignals, mRNA destabilizing sequences such as “ATTTA” [Ohme-Takagi M. etal. (1993) “The effect of sequences with high AU content on mRNAstability in tobacco,” Proc. Natl. Acad. Sci. USA 90:11811-11815], “DST”sequences [Newman TC and Ohme-Takagi M, (1993) “DST sequences, highlyconserved among plant SAUR genes, target reporter transcripts for rapiddecay in tobacco” Plant Cell 5:701-714], and the cytosine methylationmotif “CCGG”. Plant-optimized genes were designed that useplant-preferred codons and lack the potential problem sequences. Thedesigned genes for StxB and SEB were then assembled from overlappingoligonucleotides using the method described by Stemmer et al. [Stemmer WP C, et al. (1995) “Single-step assembly of a gene and entire plasmidfrom large numbers of oligodexoyribonucleotides,” Gene 164:49-53]. Thesynthetic genes were cloned and sequenced to verify the desirednucleotide sequence.

Example 3 Assembly of StxB and SEB Genes into Expression Cassette withEnhanced 35S Promoter and TEV Leader; transfer into pSK-BDY1.4H Vectorand Transformation of Tobacco

[0287] The verified synthetic StxB and SEB genes were cloned into theplant expression cassette pIBT210 [Haq TA, et al. (1995) “Oralimmunization with a recombinant bacterial antigen produced in transgenicplants,” Science 268:714-716], where they are flanked 5′ by the enhancedCaMV 35S promoter and TEV leader. The HindIII/SacI fragments from theseconstructs, containing the enhanced CaMV 35S promoter and TEV leaderfused to the coding sequence, are ligated separately with the BeYDVelements from a modified pSK-BYD1.4 [Liu L, et al. (1998) “Mutationalanalysis of bean yellow dwarf virus, a geminivirus of the genusMastrevirus that is adapted to dicotyledonous plants,” J. Gen. Virol.79:2265-2274]. Plasmid pSK-BYD1.4 is modified by filling the HindIIIsite within the rep gene and re-ligating to introduce a frameshift inthe rep coding sequence, and then introducing a HindIII site within themovement protein gene by site-directed mutagenesis, to form pSK-BDY1.4H.Digestion of pSK-BDY1.4H with HindIII and Sac removes the coat proteingene and part of the movement protein gene, and ligation of theHindIII/SacI fragments described above (35S-TEV-StxB or 35S-TEV-SEB)causes fusion of the cassettes within the BeYDV replicon such that thecoat protein gene polyadenylation signal downstream of the SacI site isutilized. The recombinant replicons are then removed from theseconstructs using SpeI digestion, and these fragments are ligated intopBin19 digested with XbaI to yield T-DNA vectors for use withAgrobacterium. These T-DNA constricts in Agrobacterium are used totransform tobacco as described [Mason H, et al (1993) “Identification ofa methyl jasmonate responsive domain in the soybean vspB promoter,”Plant Cell 5:241-251].

Example 4 Transformation of Tomato with Tandem Repeat of Mutant ViralGenome

[0288] Transgenic tomatoes containing a tandemly repeated copy (partialdimer) of the viral genome, which contains a marker gene (GUS) in placeof the viral coat protein gene (FIG. 3A), are produced. In this case,transcription of GUS is driven by the BeYDV coat protein promoter. Inanother application, the coat protein gene can be replaced by an entireexpression cassette containing GUS operably linked 5′ to a promoter suchas CaMV 35S and 3′ to a polyadenylation signal such as soybean vsp. Therep gene is inactivated by site-directed mutagenesis. The transgenicplants generated therefore contain a chromosomally integrated,replication-defective and GUS-encoding copy of the viral genome.Initially, superinfection of the transgenic plants as a model system isused to establish whether tissue specific expression of the viral repprotein will induce amplification of expression of transgenes linked tothe viral origin of replication. The wild type virus can be tested forits ability to rescue and amplify an integrated copy of a disarmed viralgenome by infecting the transgenic plants with wild type BeYDV usingeither agroinfection or biolistic inoculation of virus DNA. The infectedplants are monitored for appearance of viral symptoms. Amplification ofthe integrated viral genome in infected plants is monitored by Southernhybridization, and the expression of the GUS gene is monitored by thestandard fluorometric assay (Jefferson et al., 1987).

Example 5 Demonstration of Fruit-Specific and Ripening DependentExpression BeYDV Rep Protein in Transgenic Tomato Plants, Using the E8Promoter

[0289] Tissue-specific expression of Rep is preferred, because it may betoxic to plant cells and interfere with growth and development ifconstitutively expressed. Transgenic tomato plants are thereforeproduced that contain the BeYDV rep gene under the control of the E8promoter, which is known to be strongly expressed during fruit ripeningand is shut off in vegetative tissues. These plants are grown tomaturity and tested for rep expression in different tissues at differentdevelopmental stages by Northern blot. The activity of the Rep proteinin ripening fruit is demonstrated by biolistic delivery of thereplication-defective GUS-encoding replicon from Example 4, andreplication is monitored by Southern blot and GUS assay.

Example 6 Transgenic Tomato Plants with the E8-Rep Cassette and aReporter Gene (GUS) Expression Cassette Linked to a BeYDV Replicon

[0290] Transgenic tomato plants that contain a viral replicon (with therep gene inactivated) linked to a GUS-encoding cassette are produced.These plants are sexually crossed with the E8-rep plants from Example 5to obtain plants transgenic for both cassettes. Double transgenic linesin the progeny are selected by PCR and Southern blot of genomic DNA.When these plants are grown to maturity, expression of the Rep proteinduring fruit ripening is expected to result in rescue of the recombinantviral replicon from the chromosome in fruit tissues, and enhancedexpression of the linked GUS gene should be observed.

Example 7 Demonstration of Fruit-Specific and Ripening DependentEnhancement of Expression of GUS in Transgenic Tomato

[0291] The double transgenic lines generated in Example 6 are probed byNorthern blot to verify fruit-specific and ripening-dependent expressionof rep. GUS activity is examined at different stages of fruit ripening,and the results are compared to the GUS-only lines of Example 6 todetermine the level of enhancement of expression mediated byco-expression of rep.

Example 8 Demonstration of Seed-Specific and Ripening DependentExpression of BeYDV Rep Protein in Transgenic Soybean Plants Using theGlycinin Gy1 Promoter

[0292] The production of large amounts of a protein of interest is morepractical in a crop like soybean, where substantial amounts of proteinare stored in seeds. It is important to show seed-specific rep geneexpression and its ability to amplify a gene of interest-BeYDV repliconin seeds of soybean.

[0293] An expression cassette is constructed with the BeYDV repexpression driven by a soybean seed storage protein (glycinin) Gy1promoter (Iida et al., 1995), linked to a hygromycin selectable marker(Stewart et al., 1996). This construct is used to produce transgenicsoybean lines by biolistic DNA delivery (Christou et al., 1990; Stewartet al., 1996). A soybean cultivar with high embryogenic capacity,Glycine max L. Merrill “Jack” is used in order to facilitatetransformation. Embryogenic cultures are induced by plating immaturecotyledons on a Murashige and Skoog-based medium supplemented with 40mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogemiccultures are maintained in liquid medium containing 5 mg/liter 2,4-D.These cultures are used for microprojectile delivery of thetransformation construct. The explants are then cultured for selectionon 25 mg/liter hygromycin. Resistant embryogenic cell lines aretransferred to growth regulator-free medium to permit recovery of maturesomatic embryos. After a desiccation period, the somatic embryos arereturned to growth regulator-free medium for conversion into plants. Thetransformed lines are examined for foreign DNA copy number and integrityby PCR and Southern blot. Transformants are grown to maturity to verifyseed specific expression of Rep, after extraction of proteins asdescribed (Vitale et al., 1984). It is important to determine whetherthe seeds produced in these plants are viable, so that the plants can beused as parental lines in sexual crosses; however, it is possible toproduce double transgenic lines by incorporating two different cassettesinto a single vector plasmid.

Example 9 Production of Transgenic Soybean Plants with a Model VaccineAntigen Expression Cassette Linked to a BeYDV Replicon

[0294] A tandemly repeated (partial dimer) BeYDV replicon containing theNorwalk virus capsid protein gene (NVCP) (Mason et al., 1996) in placeof the BeYDV capsid gene is constructed, as described in Example 4 forthe GUS-containing replicon. This DNA is used to transform soybeanplants by biolistic DNA delivery (Stewart et al., 1996). Those plantscontaining a single copy of the replicon using PCR and Southern blotanalysis are selected.

Example 10 Sexual crossing of Transgenic Soybean Lines

[0295] The soybean lines obtained in Examples 8 and 9 are sexuallycrossed and seed-specific enhancement of expression of NVCP isdemonstrated. The NVCP-BeYDV plants are grown to maturity and sexuallycrossed with the Rep-expressing plants described in Example 8. Progenyplants are screened for chromosomal copies of the NVCP replicon and theRep gene cassette by PCR and Southern blot. Lines containing both of theforeign DNAs or the NVCP replicon only can be grown to maturity tocompare expression of NVCP in seeds by ELISA as described elsewhere(Mason et al., 1996).

Example 11 Transformation of Potato Plants with A. tumefaciens

[0296] With potato, it is preferred to express the rep gene with a tuberspecific promoter such as patatin or gbss. In plants containing anintegrated rep-deficient BeYDV replicon, initiation of rep expression intubers will promote rescue and replication of the integrated replicon.The stable transformation of potato (Solanum tuberosum “Frito-Lay 1607”)is achieved by axenic leaf disc co-cultivation as described elsewhere[Haq, T. et al (1995) Science, 268:714-716; Wenzler, H. et al. (1989),Plant Science 63: 79-85].

[0297] Precultivation—explants are cut and put abaxial side up on LCIplates for 3-4 days;

[0298] Inoculation—explants are placed in agro dilution for 10 minutes(agitation by hand), then blotted on sterile paper towel;

[0299] Cocultivation—after blotting, explants are put abaxial side upback on LCI plates for 3-4 days

[0300] Selection: Callus induction—after cocultivation, explants are puton LC1CK plates containing kanamycin and carbenicillin for 5-7 daysuntil callus is developed;

[0301] Shooting—explants with well developed callus are placed in LC2CKboxes for 2 weeks; after 2 weeks explants are placed on fresh LC2CKmedia and shoots are developed.

[0302] When the shoots are about 1.5 cm in length, they should betransferred to cm media for root formation. Carbenicillin is added to cmmedia to prevent bacterial growth in excess.

[0303] Growing Agrobacterium—LB stock: one colony is cultured overnightin presence of kanamycin; YM stock: one colony is cultured over a 3648hour period in the presence of kanamycin.

[0304] Because some variation in expression among independenttransformation events is expected due to random site of insertion,independent transgenic lines are screened by Northern blot using codingregion-specific probes. When a 35S promoter is used, the total RNA fromleaves is screened. When the patatin promoter is used, RNA is obtainedfrom microtubers developed in axenic culture. The patatin promoterconstructs are screened from microtubers generated by subculture of stemnode cuttings from nascent transformed shoots on media high in sucrose.The most promising transformed lines are propagated by axenic culture ofnode cuttings; and rooted plantlets are transplanted to soil and grownin the greenhouse.

[0305] To obtain tubers, the plants are grown in a two-stage protocol:(1) for the first 1.5-2 months, or until sufficient vegetative growthdevelops, using long day (16 h) lighting with supplemental lighting inwinter, and (2) for an additional 1.5-2 months under short day (12 h)conditions. The tubers are typically recovered in yields of 600-800 gper 3-gallon pot after this 3-4 month regime. Tubers harvested from thefirst crop can be used to initiate new plants after a 1-month dormancyperiod.

Example 12 Transformation of Tomato Plants

[0306] (as modified from Frary/Fillati et al. (1987) Biotechnology5:726-730)

[0307] Preparation of Plant Material:

[0308] Sterilize Seed

[0309] a.) Immerse seed (Tanksley TA234TM2R) in 20% CHLOROX bleach for20 min. (100 seeds˜380 mg)

[0310] b.) Rinse well with sterile milli-Q water (2 or more times.)

[0311] Sow seed in Magenta boxes containing ½ MSO (approximately 30seeds/box)

[0312] Prepare Feeder Layer (One Day Prior to Cutting Cotyledons).

[0313] a.) Pipet 2 mL of a one week old NT-1 suspension culture onto aKCMS media plate. (NT-1 cells are subcultured weekly (2:48) in KCMSliquid medium.)

[0314] b.) Cover suspension with a sterile 7 mc. Whatman filter

[0315] c.) Culture in dark, overnight

[0316] 4.) Cut cotyledons 8 days after sowing.

[0317] Place seedling on a sterile paper towel moistened with sterilewater

[0318] Excise cotyledon at petiole and cut tips off. Cut in half againif size of cotyledon is >1 cm.

[0319] c.) Place explants on feeder plates adaxial side down

[0320] d.) Culture 25° C., 16 hour photoperiod, overnight

[0321] Transformation:

[0322] 1.) Streak Agrobacterium onto LB selective media plate about 1week prior to transformation. Incubate at 30° C.

[0323] 2.) Inoculate liquid selective medium.

[0324] Pick a single colony off of streaked plate, put into 3 mL YMmedium with 150 μg Kanamycin Sulfate. Shake vigorously at 30° C. for 48hours.

[0325] Pipette 1 mL of inoculate into 49 mL YM medium with 2.5 mgKanamycin Sulfate and culture in 250 mL flask shaking vigorously at 30°C. for 24 h.

[0326]  Measure O.D. of Agro culture using a spectrophotometer at 600nm. Optimum O.D. equals 0.5 to 0.6.

[0327]  Prepare Agro culture for transformation.

[0328] Centrifuge Agro culture at 8,000 rpm (Sorvall centrifuge, ss34rotor) for 10 min.

[0329] Pour off YM and resuspend in MS-O, 2%. Final O.D. should bebetween 0.5 and 0.6.

[0330] 5.) Incubate explants in Agro culture/MS-0,2%

[0331] a.) Pipette 25 mL of Agro culture into a sterile Magenta box.

[0332] b.) Transfer explants from 2 to 3 plates into inoculum in magentabox.

[0333] c.) Incubate for 5 min. with occasional shaking.

[0334] d.) Remove explants to a sterile paper towel.

[0335] e.) Return explants to feeder plates, adaxial side down.

[0336] Seal plates with Nesco film.

[0337] 6.) Cocultivate explants, 16-hour photoperiod, 25° C. , forapproximately 24 h.

[0338] Transfer explants to selection media (2Z) adaxial side up. Sealplates with micropore tape. Return to 25° C., 16-hour photoperiod.

[0339] Transfer explants to new IZ selection medium plates every 3weeks. When shoots begin to appear transfer to IZ Magenta boxes.

[0340] Regeneration/Rooting:

[0341] Within 4 to 6 weeks initial shoots should appear.

[0342] Excise shoots from explants when shoots are at least 2 cm. andinclude at least 1 node. Place in Magenta boxes (4/box) containingTomato Rooting Media with selective agents.

[0343] Roots should begin to appear in about 2 weeks.

Example 13 Regeneration of Transgenic Tomatoes

[0344] Standard Greenhouse Growth Conditions:

[0345] 16 hour day

[0346] average Temperature: 24.5° C.

[0347] fertilized each time watered: 100 ppm. EXCELL (15-5-15) withextra Calcium and Magnesium

[0348] Potatoes grown in METRO-MIX 360

[0349] Tomatoes grown in Cornell Mix +OSMO

[0350] Biological controls are used whenever possible to improve overallplant quality

[0351] The present invention has been described with reference toparticular examples for purposes of clarity and understanding. It shouldbe appreciated that certain improvements and modifications of theinvention can be practiced within the scope of the appended claims andequivalents thereto.

Example 14 Creation of Transgenic Plants with pBeYDV1.4/Bin19

[0352] Transgenic tobacco (Nicotiana benthamiana, Nicotiana tabacum)plant lines were created using the BeYDV genome in the vectorpBeYDV1.4/Bin19 (Liu et al., supra) according to the methods describedabove, in order to demonstrate that an integrated viral genome can beexcised and replicated.

[0353] Some of these transgenic tobacco plants displayed a stuntyphenotype suggesting virus infection, while other transgenic tobaccolines appeared normal. Southern blot analysis of plant leaf DNA probedwith BeYDV genomic DNA demonstrates the presence of replicative forms ofBeYDV DNA in the stunty plants, and the absence of replicative forms innormal plants or plants transformed with the vector alone (data notshown). These data demonstrate that integrated BeYDV genomic DNA can berescued and replicated in some transgenic lines.

[0354] In the nonreplicating transgenic lines, the site of insertioninto the plant chromosomal DNA may limit expression of the Rep protein,which is needed for excision and replication of the viral DNA.

[0355] Further, electron microscopy of thin sections of leaves fromtransgenic lines demonstrating a stunty phenotype revealed the presenceof crystalline arrays of virions characteristic of geminivirus-infectedplants. Negatively stained leaf extracts showed geminate virus particles(data not shown).

Example 15 Use of BeYDV Cassettes in Transient Assays with Tobacco Cells

[0356] To produce transgenic plants expressing the Rep gene in trans, aseries of constructs containing the Rep gene under constitutive (35S),inducible (AlcA) and developmental (E8) promoters were prepared.

[0357] It has been demonstrated that removal of the intron within theRep gene greatly enhances BeYDV replication. Therefore, a series ofclones containing this intronless form of Rep (Δintron) were alsoconstructed. Reporter gene cassettes and Rep expression cassettes wereconstructed to test the replication of recombinant replicons in tobaccocells (FIG. 26).

[0358] Preliminary assessment of the ability of the BYDV based Greenfluorescent protein (GFP) expression construct to replicate in-vivo whensupplied with a Rep protein was tested by transient assays in tobaccoNT-1 cell cultures. The GFP cassette was introduced into the cells byparticle bombardment, either alone or in the presence of theself-replicating wild-type virus (FIG. 26).

[0359] Two days after bombardment, a high proportion of the cells incultures bombarded with the GFP construct alone or with both constructs,exhibited a high level of GFP fluorescence (data not shown). Thefluorescent signal in the cells bombarded with the GFP construct alonedecreased rapidly as evident by a low number of residual cells thatexhibited fluorescence and a low level of fluorescence after four days(data not shown). Six days after the bombardment, GFP could not bevisualized in these cultures (data not shown). However, a high level ofGFP was maintained in cultures of cells bombarded with both thereplicating reporter construct and the Rep producing construct for atleast 6 days (FIGS. 24b and 24 c). These results demonstrate replicationof the GFP construct in the presence of trans-acting Rep protein, butnot in the absence of Rep.

[0360] Primers designed in opposing orientations (GFP5′:5′-AGCTCGACCAGGATGG and GFP3′: 5′-GTCCTGCTGGAGTTCG) were used todetermine whether Rep expression in trans is capable of promotingnicking and religation of the GFP expression cassette, indicative of theoccurrence of Rep-mediated replication. PCR products of the predicted 2kb size were detected from total DNA isolated from cells bombarded withthe GFP cassette in the presence of any of three different Rep producingplasmids that produce Rep, but not from DNA isolated from cellsbombarded with the GFP cassette alone (data not shown). These dataconfirm that a Rep protein provided in trans can excise and replicate areplicon present on a different segment of DNA.

[0361] To gain a more quantitative estimate of the level of reportergene expression from different constructs, a series of Rep constructsand GUS reporter cassettes were designed and cobombarded into NT-1cells. Relative GUS activity was determined (FIG. 27). In FIG. 27, GUSexpression is shown as a function of days after transfection wherein aGUS cassette was delivered with wild type virus (closed diamonds),35SΔintron (X), REP+MP-CP-(closed squares), 35SRep (closed triangles)and 35SfsRep(*).

[0362] One week post-bombardment, GUS activity from cells cobombardedwith both wild-type virus and the GUS expression cassette was found tobe 8 times higher than cells bombarded with the GUS expression cassettealone. Similarly, GUS activity was found to be several fold higher incells cobombarded with the expression cassette in the presence of the35SRep construct, the Δintron constructs, or the Rep+MP-CP-construct ascompared to cells bombarded with the GUS expression cassette alone. NoGUS activity could be detected from cells cob ombarded with a frameshiftversion of 35Srep (35SfsRep) or from a GUS cassette containing an LIRbut lacking an SIR (data not shown).

[0363] Over a three week period GUS activity was greatest in cellscobombarded with wild type virus; GUS levels increased for two weeks,then slowly declined, most likely due to the age of the cells. GUSactivity in cells transfected with 35SRep and 35SΔintron was comparableto that obtained with the Rep+MP-CP-construct. In all cases, levels weregreatest by 1 week post cobombardment and were maintained, demonstratingonly a moderate decrease for the next two weeks. Cells cobombarded with35SfsRep and the reporter cassette expressed GUS transiently at lowlevels for the first 4 days; by one week post-cobombardment, thisactivity was lost completely.

Example 16 Replication of a BYDV Based GFP Expression Construct in thePresence of a Rep Protein Producing Construct

[0364] Construction of a Replicatable Expression Cassette.

[0365] The PstI-BamHI fragment containing the long intergenic region(LIR) of the Bean Yellow Dwarf Virus (BeYDV, cloned as a partial dimerin pBluescript (Stratagene), BY002. See FIG. 4) was cloned intopBlueScriptKS (Stratagene) to give pBY017 (FIG. 4). A fragmentcontaining the short intergenic region (SIR) of BYDV was PCR amplifiedfrom the same plasmid using the primers SIRfor and SIRrev (see box 1,below) and cloned into pBY017 by replacing the short HindIII-EcoRIfragment to give BY019 (FIG. 4). The NheI-BamHI fragment of this plasmidthat contains the LIR and the SIR, was cloned into pBY017 replacing theXbaI-BamHI fragment thus yielding pBY020 (FIG. 4). The insert thatcontains a tandem array of LIR-SIR-LIR was amplified from pBY020 by atwo-step PCR by using the primers LIRAscI and SIRrev (Box 1, below). Theresultant PCR fragment was used as a megaprimer together with the primerLIRFseI (Box 1, below) to amplify the fill length LIR-SIR-LIR, nowflanked by sites for the 8-base restrictases, AscI and FseI. Thisfragment was then cloned into a pUC19 derivative (pBY022, not shown),the multiple cloning region of which was replaced by a linker containingthe restriction sites for AscI, FseI and PmeI. The resultant plasmid,pBY024 (FIG. 4), was sequenced and will serve as an intermediate vectorfor cloning of genes of interest between the PstI site and the EcoRIsites. The replicatable expression cassette is excised with AscI andFseI and cloned into a binary vector, a derivative of pGPTV-Kan (Beckeret al., 1992, Plant Mol. Biol., 20:1195) in which the HindIII-EcoRIfragment was replaced by the same linker described above (pBY023, notshown).

[0366] Box 1. Oligonucleotides used in this study. Name: Sequence SIRfor5′-GGAATTCCGAGTGTACTTCAAGTCAGTTGG-3′ SIRrev5′-GGAAGCTTGGGATCCCTTCTATAATTCTTTGG-3′ LIRAscI5′-TGGCGCGCCGCTCTAGCAGAAGGCATGTTG-3′ LIRFseI5′-TTGGCCGGCCGTACGAATAATTCGTATCCGAC-3′ LinkUP5′-AGCTGGCGCGCCGTTTAAACGGCCGGCC-3′ LinkDown5′-TTAACCGGCCGGCAAATTTGCCGCGCGG-3′

[0367] Construction of Reporter Constructs

[0368] Two reporter gene expression cassettes were inserted into thereplicatable pBY024 plasmid described above. First, the gene encodingthe green fluorescence protein (GFP) containing the TEV-5′UTR and thenos-3′UTR, under the control of the Cauliflower Mosaic Virus 35Spromoter with double enhancer (35S) was cloned between the PstI andEcoRI restriction sites of pBY024 to yield pBY027 (FIG. 4). Thisconstruct was used in a transient expression study to verify itsreplicatability (see below). Another reporter gene, UidA (under thecontrol of the 35S promoter with the TEV-5′UTR and the 35S-3′UTR) wascloned into the PstI site (pBY028, FIG. 4). This construct is used toproduce more quantifiable assessments (by the GUS assay) of the level ofexpression with the gemivirus construct. These constructs are clonedinto binary vectors (by methods well known in the art) for the creationof transgenic plants.

[0369] Replication of the Plasmid pBY027 by an Infectious Clone of BYDV.

[0370] Preliminary assessment of the BYDV based GFP expressionconstruct, pBY027, to replicate in-vivo when supplied with thereplication associated protein of BYDV (Rep/RepA) was tested bytransient assays in NT-1 cell cultures. The plasmid pBY027 wasintroduced into the cells by particle bombardment, either alone or withthe self-replicating BYDV construct pBY003 (alias pBeYDV1.4/Bin19, Liuet al., supra).

[0371] Two days after bombardment, a high proportion of the cells inculture that were bombarded with the pBY027 alone or with both plasmids,exhibited a high level of GFP expression (data not shown). The levels ofGFP in the cells bombarded with the expression construct alone decreasedrapidly as demonstrated by the residual number of cells that exhibitedfluorescence and the level of the fluorescence after four days (FIG.27a). Six days after the bombardment, GFP could not be visualized inthese cultures (data not shown). However, high levels of GFP wasmaintained in cultures of cells bombarded with both the replicatingreporter construct and the Rep/RepA producing construct for at least 6days (FIGS. 27b and 27 c). These results indicate that replication ofthe reporter construct occurs in the presence of trans-acting Repprotein and not in its absence.

Example 17 Production of Transgenic Plants with SEB-F44S Gene

[0372] An expression cassette for the plant-optimized SEB-F44S geneunder the control of the constitutive 35S promoter was constructed (FIG.28), and used to transform potato. The expression cassette for SEB-F44Sis driven by the constitutive Cauliflower Mosaic Virus 35S promoterlinked to the Tobacco Etch Virus 5′-UTR, and flanked 3′ by the soybeanvspB 3′ element.

[0373] Plants are regenerated on selective medium and screened for SEBexpression by ELISA two weeks later when the potato plantlets are largeenough. An ELISA assay is carried out in the presence of a positivecontrol, SEB obtained from SIGMA. Selected lines are grown to maturityin the greenhouse.

Example 18 Construction and Testing of a BeYDV Construct

[0374] A BeYDV construct for insertion of expression cassettes that canform replication-competent episomes was constructed. This BeYDVconstruct contains 2 copies of the long intergenic region (LIR) flankingthe short intergenic region (SIR) and a polylinker site for insertion ofexpression cassettes (FIG. 5, pBY024). We have inserted cassettes forexpression of SEB-F44S (FIG. 28), hepatitis B surface antigen (HbsAg)(pBY217, FIG. 20), and reporter genes GUS (pBY028, FIG. 8 and pBY029,FIG. 9) and GFP (pBY027, FIG. 7).

[0375] The HBsAg construct was tested by codelivery to tobacco NT1 cellsin the presence or absence of pBY002 (FIG. 5) as a source of Repexpression. Cells were incubated either for 3 days or for 6 days. Thecellular DNA of the transformed tobacco cells was analyzed by Southernblot (data not shown) either undigested (U) or after digestion withBamHI (B) or DpnI (D). The Southern blot was probed with a ³²P-labeledHBsAg sequence.

[0376] Undigested DNA isolated from cells transformed with pBY217 andpBY002 shows a strong signal at 2.3 kb that is entirely shifted to 3.0kb after digestion with BamHI, which site is unique in pBY217. SincepBY217 is approx. 6.0 kb, the data shows that the cassette was excisedand replicated as the 3.0 kb size expected. Cells transformed withpBY217 alone showed no signal, indicating that the plasmid concentrationwas too low to detect (data not shown). These data demonstrate that aconstruct comprising two copies of the long intergenic region (LIR)flanking the short intergenic region (SIR) and a polylinker site forinsertion of expression cassette can be replicated when Rep is providedin trans.

Example 19 Production of Polyclonal Antiserum Against Rep and Detectionof Rep During BeYDV Infection or Transient Expression in NT1 Cells

[0377] Generation of Antiserum Against BeYDV Rep

[0378] An intronless form of the Rep gene in pΔintron (Liu et al.,supra) was PCR amplified using the primers 5NcoRep CGG ATA ACA ATT TCACAC AG and 3′ BglIIRep CTC AGC TAA TTA AGC TTA, and cloned into the NcoIand BglII sites of the plasmid QE60 (Qiagen) to produce the constructQE60Rep. This 6His-tagged version of Rep was purified using the standardQiagen QIAExpress Kit and injected subcutaneously into a rabbit in aseries of four doses of 0.5 mg of purified protein in PBS. The firstdose was emulsified in Freund's complete adjuvant, followed 5 weekslater by a second dose in Freund's incomplete adjuvant, and third andfourth doses in Freund's incomplete adjuvant at 3-week intervals. Theserum was obtain 3 weeks after the last dose and stored at −20° C.

[0379] Detection of Rep During BeYDV Infection or Transient Expressionin NT1 Cells

[0380] Tobacco NT1 cells were bombarded with various plasmids includingpBY002 (native virus), paintron (virus with C1/C2 intron deleted),p35SRep, p35Sdintron, and p35SRepA. At 2, 4, 6, and 8 days after DNAdelivery, cells were extracted and assayed by Western blot using therabbit polyclonal antiserum against Rep, diluted at 1:1000. Low amountsof Rep and RepA was detected in cells bombarded with wild type virus DNA(pBY002). A similar expression pattern was observed when Rep was placedunder the control of the 35S promoter (p35SRep), although levels weresomewhat higher. The amount of Rep protein produced from an intronlessconstruct (pΔintron) was higher than the amount of protein produced by awild type construct and an even greater amount of Rep protein wasproduced from a construct wherein the intronless rep gene is under thecontrol of the promoter 35S (p35SΔintron) (data not shown). These datademonstrate that the antiserum against Rep is a valuable reagent fordetermination of Rep expression and the relative levels of Rep and RepAin plant tissues.

Example 20 Use of BeYDV “LIR-SIR-LIR” Replicons to Enhance ProteinExpression in the Presence of Alcohol-Inducible Expression of RepProtein

[0381] This example describes the use of a BeYDV LIR-SIR-LIR (LSL)replicon (a derivative of pBY024) to achieve amplification of thecassette only in the tissues of plants that are treated with a chemicalinducer of Rep gene expression.

[0382] The binary T-DNA plasmid vector pBYSEB-410 (FIG. 29) contains anexpression cassette for SEB-F44S in a replication-competent context(i.e., linked to an SIR element and flanked by both the duplicated LIRregions of BeYDV) and a (bialaphos resistance gene) BAR selectionmarker. Thus pBYSEB-410 is used to transform Arabidopsis thaliana usingthe method of A. tumefaciens-mediated DNA delivery to flowers as(described in Clough and Bent, supra).

[0383] The binary T-DNA plasmid vector pSRN-ADP(F) contains anexpression cassette for Rep-Δintron (a rep gene with the intron deleted)under the control of the AlcA alcohol-inducible promoter, thealcohol-responsive AlcR gene driven by the constitutive 35S promoter,and a NptII selection marker. Thus pSRN-ADP(F) (FIG. 23) is used totransform A. thaliana using the method of A. tumefaciens-mediated DNAdelivery to flowers. Transformation of flowers using both vectors can beachieved simultaneously using two A. tumefaciens-stains transformed witheither plasmid.

[0384] Progeny seeds are germinated and selected on media containing 50mg/L kanamycin and 1 mg/L phosphinothricin, in order to obtain plantsthat contain both the NptII and BAR selection markers. Southern blotanalysis or PCR analysis with gene-specific primers is used to confirmthe presence of the foreign DNA from either vector in the progeny.Plants that are demonstrated to be transgenic for both pBYSEB-410 andpSRN-ADP(F) are propagated and grown in soil. Seeds are also selected on1 mg/L phosphinothricin alone in order to obtain control plantstransgenic only for pBYSEB-410. After selection for the BAR marker, leafexplants are tested for kanamycin sensitivity to identify plantstransgenic only for pBYSEB-410 and not for pSRN-ADP(F).

[0385] In order to induce the AlcA promoter controlling Rep expression,the plants are watered with a solution of 1% ethanol. Leaf tissues aresampled on successive days after ethanol induction to verify appropriateexpression of Rep using Western blot and SEB expression by ELISA orWestern blot. Copy number of the replicating episome is examined bySouthern blot. It is expected that the SEB expression will be at least10-fold higher in the pBYSEB-410/pSRN-ADP(F) plants than in thepBYSEB-410 plants.

[0386] Other dicotyledon plants are also co-transformed with pBYSEB-410(or similar vectors containing alternative expression cassettes,described herein) and pSRN-ADP(F). For example, potato plants are usedas hosts, and the resulting tubers of double transgenic lines aretreated with ethanol to induce Rep expression and thus replication ofthe BeYDV LSL replicon.

Example 21 Use of BeYDV LSL Replicons to Enhance Expression in aFruit-Specific Manner

[0387] This example describes the use of a BeYDV “LSL” replicon(derivative of pBY024) to achieve amplification of the cassette only inripening fruit tissues of tomato. The binary T-DNA plasmid vector pBY031contains a GUS expression cassette in a replication-competent context(i.e., linked to a SIR element and between the duplicated LIR regions ofBeYDV). Thus pBY031 is used to transform tomato (Lycopersiconesculentum) using A. tumefaciens-mediated DNA delivery. Transformedplants expressing GUS in the fruit are selected by histochemical stainfor GUS or by fluorometric assay for GUS (as described in Jefferson,1987, Plant Mol. Biol. Report, 5:387) and seeds are obtained, washed,and dried. To measure expression of other proteins donor. Since thematernal tissues comprise the fruit, the fruit of pBY031/pE8-Rep plantsshould behave similarly with regard to GUS expression regardless of thepollen donor. pBY031 plants are grown separately as baseline controls.During fruit ripening, as the color of the fruit begins to change fromgreen to yellow, orange, and then red, the E8 promoter will become moreand more active, driving transcription of the Rep gene and leading toexpression of the Rep protein. Fruits are sampled at different ripeningstages to verify appropriate expression of Rep using Western blot and toexamine GUS expression by enzymatic assay. Copy number of thereplicating episome is examined by Southern blot. It is expected thatthe GUS expression will be at least 10-fold higher in the pBY031/pE8-Repfruits than in the pBY031 fruits.

[0388] An alternative to the sexual crossing strategy to obtainpBY031/pE8-Rep plants is as follows. A binary T-DNA vector that containsthe cassettes for both the GUS/LSL and the E8-Rep linked between rightand left T-DNA border elements is constructed. This construct is used totransform plants and examine episomal replication and enhancement ofexpression in the primary transformed lines.

[0389] Such a system can be used in other dicotyledon plants byregulating the expression of RepC1/C2 or Rep-Δintron with a homologousfruit ripening-specific promoter.

Example 22 Use of BeYDV “LSL” Replicons to Enhance Protein Expression inSeeds

[0390] This example describes the use of a BeYDV “LSL” replicon(derivative of pBY024) to achieve amplification of the cassette only inseeds of Arabidopsis thaliana plants. The binary T-DNA plasmid vectorpBYSEB-410 (FIG. 29) contains an expression cassette for SEB-F44S in areplication-competent context (i.e., linked to a SIR element and betweenthe duplicated LIR regions of BeYDV) and a BAR selection marker. Thebinary T-DNA plasmid vector p2S-Δintron contains an expression cassettefor Rep-Δintron (with intron deleted) under the control of the A.thaliana 2S2 seed protein promoter and a NptII selection marker. ThuspBYSEB-410 and p2S-Δintron are used to transform A. thaliana using A.tumefaciens-mediated DNA delivery to flowers. Transformation of flowersusing both vectors can be achieved simultaneously using two A.tumefaciens-stains transformed with either plasmid.

[0391] Progeny seeds are then germinated and selected on mediacontaining 50 mg/L kanamycin and 1 mg/L phosphinothricin, in order toobtain plants that carry both the NptII and BAR selection markers.Southern blot or PCR with gene-specific primers is used to confirm thepresence of the foreign DNA from either vector in the progeny. Plantsthat are confirmed transgenic for both pBYSEB-410 and p2S-Δintron arepropagated and grown in soil. Seeds are also selected on 1 mg/Lphosphinothricin alone in order to obtain control plants transgenic onlyfor pBYSEB-410. After selection for the BAR marker, leaf explants aretested for kanamycin sensitivity to identify plants transgenic only forpBYSEB-410 and not for p2S-Δintron.

[0392] Plants are grown to maturity and during seed maturation, theseeds are extracted to verify appropriate expression of Rep usingWestern blot analysis and to examine SEB expression by ELISA or Westernblot analysis. Copy number of the replicating episome is examined bySouthern blot analysis. It is expected that the SEB expression will beat least 10-fold higher in the pBYSEB-410/p2S-Δintron plants than in thepBYSEB-410 plants.

[0393] Other dicotyledon plants are co-transformed with pBYSEB-410 (orsimilar vectors containing alternative expression cassettes) andp2S-Δintron. For example, soybean plants are used as hosts, and Repexpression is controlled by fusion to a glycinin seed specific promoter(Iida et al., 1995, Plant Cell Reports, 14: 539).

Example 23 Enhancement of Expression of GUS Using a Replicating BeYDV“LSL” Cassette

[0394] This experiment demonstrates that transient expression of GUS intobacco NT-1 cells can be enhanced greatly by inclusion of the GUSexpression cassette in the “LSL” cassette exemplified in pBY024.

[0395] Mid-logarithmic NT-1 cell cultures (about 10⁶ cells/ml) were letto settle and resuspended in half of the original volume of culturemedium. One ml of this suspension was plated on NT solid medium inpatches of about 2.5 cm in diameter. Gold particles (1 μm, BioRad) werecoated with plasmid DNA as described by Sanford et al. (Methods inEnzymol. 1993; 217, 483-509). Each plate was bombarded by gold particleparticles coated with 0.25 nmol plasmid DNA (in case of co-delivery,0.25 nmol of each of the two constructs) of either pBY028, pBY029 withor without pBY002. On days three and six after bombardment half of thecell mass from three different plate was harvested and total solubleproteins were extracted as described by Jefferson (Jefferson, Plant Mol.Biol. Report. 1987; 5:387). GUS activity was assayed according toJefferson (supra). The results demonstrated in FIG. 31 clearly show thatfor pBY028+pBY002, GUS activity at 3 days after bombardment isapproximately 50 to 100 times higher than for pBY028 alone. At 6 daysafter bombardment, the enhancement is less striking, but still 15 to 30fold higher than for pBY028 alone. It is likely that the enhancedexpression is a result of amplification of the expression cassette whenpBY002 is present to provide Rep protein to mediate the excision andreplication of the “LSL” replicon of pBY028. pBY029, which is similar topBY028 but wherein the GUS expression cassette is inserted in reverseorientation within the LSL cassette, showed substantially lessenhancement when co-delivered with pBY002, but still showedapproximately 3-fold enhancement at day 3. It is not clear whether thelower enhancement with pBY029+pBY002 (relative to pBY028+pBY002) is aresult of impaired replication or transcription.

Example 24 Divergent PCR Confirms Replication of LSL Constructs

[0396] Divergent PCR is used to confirm replication of a constructaccording to the invention (for example LIR-GFP-SIR-LIR). Tobacco NT1cells were bombarded with the reporter construct containing greenfluorescent protein gene (GFP) inserted between LIR and SIR, with asecond downstream LIR. When such constructs that contain duplicated LIRelements are present in cells with Rep protein, recombination betweenthe LIR elements to release the intervening DNA allows replication ofthe episome. Thus divergent primers are used to amplify the predictedsized recombinant fragment only when replication occurs. FIG. 32indicates that the predicted 2 kb DNA fragment is amplified by PCR insamples from cells co-bombarded with Rep-producing constructs, but notwith the reporter construct alone. In FIG. 32 lane 1 is a DNA ladder,lane 2 is BeYDV (pBY002), lane 3 is p35SRep, lane 4 is p35SΔintron, lane5 is cassette alone and lane 6 is a DNA ladder.

Example 25 SIR is Required for Episomal Replication

[0397] NT-1 cells were bombarded with pLIR-GUS-SIR (FIG. 33) (in thepresence or absence of a Rep protein provided by pBY002) or withpLIR-GUS (FIG. 34) (in the presence or absence of a Rep protein providedby pBY002). Southern blot analysis demonstrates that the GUS containingreplicon fails to replicate in the absence of an SIR element (data notshown).

Example 26 Use of a Chimeric Oligoniucleotide to Induce Replication ofRecombinant Genome-Integrated BeYDV Replicons

[0398] This example illustrates how a novel mechanism can be used toobtain a genetic mutation in a plant that will induce replication ofgenome-integrated BeYDV replicons. Self-complimentary chimericoligonucleotides (COs) composed of DNA and modified RNA residues havebeen used to perform gene-specific mutations in plant cells (Beetham etal., 1999, Proc. Natl. Acad. Sci. USA, 96:8774; and Zhu et al., 1999,Proc. Natl. Acad. Sci. USA, 96: 8768).

[0399] A mutated form of pBY002 (pmBY002) is constructed using theQuickChange (Stratagene) mutagenesis kit and the complimentaryoligonucleotides: 5′-CCTTGTATTAGGGTCCTTCTTTTTTCG and5′-CGAAAAAAGAAGGACCCTAATACAAGG. This mutation causes deletion of asingle “A” nucleotide within the 5′ BamHI site in the C1 portion of thecoding sequence of RepC1/C2, and thus a reading frameshift that preventsproduction of Rep. Since pBY002 contains a whole BeYDV genome andduplicated LIR elements, a reversion in the mutation described byinsertion of the deleted “A′” nucleotide would restore the infectivityof the mutant clone in pmBY002. The mutant BeYDV 1.4-mer is obtained bydigestion of pmBY002 with XbaI and partial digestion with EcoRI,inserted into pGPTV-BAR (Becker et al., 1992, Plant Mol. Biol., 20:1195)and digested with XbaI and EcoRI to form pmBY002-BAR (FIG. 35).

[0400] pmBY002-BAR is used to transform tomato (Lycopersicon esculentum)using A. tumefaciens-mediated DNA delivery. Transformed lines areselected on media containing 1 mg/L phosphinothricin, and the presenceof the transgene is confirmed by Southern blot using BeYDV sequenceprobes and PCR using BeYDV-specific primers with genomic DNA fromleaves. Positive lines are grown to maturity, and seeds are obtained,washed, and dried.

[0401] pBY031 contains a GUS expression cassette in areplication-competent context (i.e., linked to a SIR element and betweenthe duplicated LIR regions of BeYDV), and a NptII selection markerwithin T-DNA borders. Thus pBY031 is used to transform tomato(Lycopersicon esculentum) using A. tumefaciens-mediated DNA delivery.Transformed plants expressing GUS in the fruit are selected byhistochemical stain for GUS or by fluorometric assay for GUS (Jefferson,1987, Plant Mol. Biol. Report, 5:387) and seeds are obtained, washed,and dried.

[0402] The seeds of lines transgenic for pBY031, and pmBY002-BAR arestored at 4° C. for one month, and then germinated and selected onmedium containing either 50 mg/L kanamycin (pBY031) or 1 mg/Lphosphinothricin (pmBY002-BAR). Potential homozygous lines are selectedby Southern blot of genomic DNA from leaves, and candidates are grown tothe stage of flowering. The flowers are self-pollinated, and seedsobtained from ripe fruit are washed and dried. After storage at 4° C.for one month, 25 seeds from candidate homozygous lines are germinatedand selected on medium containing 50 mg/L kanamycin (pBY031) or 1 mg/Lphosphinothricin (pmBY002-BAR). Those lines that yield 100% resistantprogeny are considered homozygous.

[0403] The homozygous lines are grown to the stage of flowering, andpBY031 plants are crossed with pmBY002-BAR plants. The cross isperformed either by placing pollen from pBY031 plants on the stigma ofpmBY002-BAR plants, or by placing pollen from pmBY002-BAR plants on thestigma of pBY031 plants. In either case, 100% of the progeny shouldcarry a single-locus transgene from pBY031 and pmBY002-BAR. Seeds fromripe fruit are obtained, washed, and dried. After storage at 4° C. forone month, seeds of pBY031/pmBY002-BAR plants are germinated and plantsare grown in soil.

[0404] A chimeric oligonucleotide (CO) designed to insert the deleted“A” nucleotide into the mutated C1 sequence in pmBY002-BAR is created asdescribed (Beetham et al., supra). The CO is delivered into leaves ofeither pBY031 or pBY031/pmBY002-BAR plants by microprojectilebombardment as described (Beetham et al., supra). The mutation allowsthe BeYDV replicon to become active by acquisition of the capacity toproduce an active Rep protein. The Rep protein present in cellsharboring the virus mediates the excision and replication of the GUSreplicon in the transgene delivered by pBY031. The virus movessystemically throughout the plant, producing Rep and replicating itselfand the GUS replicon in the process.

[0405] Leaves are sampled at different times after delivery of the CO toverify appropriate expression of Rep by Western blot and GUS expressionby enzymatic assay. The copy number of the replicating episome isdetermined by Southern blot. It is expected that the GUS expression willbe at least 10-fold higher in the pBY031/pmBY002-BAR plants than in thepBY031 plants.

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1 22 1 303 DNA bean yellow dwarf virus 1 aggcatgttg ttgtgactccgaggggttgc ctcaaactct atcttataac cggcgtggag 60 gcatggaggc aagggcattttggtaattta agtagttagt ggaaaatgac gtcatttact 120 taaagacgaa gtcttgcgacaaggggggcc cacgccgaat tttaatatta ccggcgtggc 180 cccaccttat cgcgagtgctttagcacgag cggtccagat ttaaagtaga aaagttcccg 240 cccactaggg ttaaaggtgttcacactata aaagcatata cgatgtgatg gtatttgatg 300 gag 303 2 1110 DNA beanyellow dwarf virus 2 ccatgggacc ttctgctagc aagaacttca gactccaatctaaatatgtt ttccttacct 60 accccaagtg ctcatctcaa agagatgatt tattccagtttctctgggag aaactcacac 120 cttttcttat tttcttcctt ggtgttgctt ctgagcttcatcaagatggc actacccact 180 atcatgctct tatccagctt gataaaaaac cttgtattagggatccttct tttttcgatt 240 ttgaaggaaa tcaccctaat atccagccag ctagaaactctaaacaagtc cttgattaca 300 tatcaaagga cggagatatt aaaaccagag gagatttccgagatcataag gtctctcctc 360 gcaaatctga cgcacgatgg cgaactatta tccagactgcaacgtctaag gaggagtatc 420 ttgacatgat caaagaagaa ttccctcatg aatgggcaacaaagcttcaa tggctggaat 480 attcagccaa caaattattt cctccacaac ctgagcagtacgtgtcgccc ttcacagaat 540 cagatctccg ctgccacgaa gatctgcaca actggagagagacgcaccta tatcatgtaa 600 gcatcgatgc ctacactttc atacatcctg tctcctacgatcaagcacaa tctgaccttg 660 agtggatggc cgatctaacc aggatgaggg aaggactggggtcagacacc ccagcctcta 720 catctgcgga ccaactcgta ccggaaagac cacctgggctagaagtctcg ggcgacacaa 780 ctactggaac gggaccatcg acttcaccaa ctacgatgaacacgccacct ataatatcat 840 cgacgacatc cccttcaagt tcgtcccatt gtggaagcaattaataggtt gccagtctga 900 tttcactgtc aaccctaaat atggaaaaaa gaagaaaataaaaggtggga tcccttctat 960 aattctttgg aatcctgacg aagactggat gttatcaatgacaagtcaac agaaggatta 1020 ctttgaagat aattgcgtca cccactatat gtgtgacggggagacttttt ttgctcggga 1080 atcgtcgagt cactgaacgt gcctgagctc 1110 3 293PRT bean yellow dwarf virus 3 Met Gly Pro Ser Ala Ser Lys Asn Phe ArgLeu Gln Ser Lys Tyr Val 1 5 10 15 Phe Leu Thr Tyr Pro Lys Cys Ser SerGln Arg Asp Asp Leu Phe Gln 20 25 30 Phe Leu Trp Glu Lys Leu Thr Pro PheLeu Ile Phe Phe Leu Gly Val 35 40 45 Ala Ser Glu Leu His Gln Asp Gly ThrThr His Tyr His Ala Leu Ile 50 55 60 Gln Leu Asp Lys Lys Pro Cys Ile ArgAsp Pro Ser Phe Phe Asp Phe 65 70 75 80 Glu Gly Asn His Pro Asn Ile GlnPro Ala Arg Asn Ser Lys Gln Val 85 90 95 Leu Asp Tyr Ile Ser Lys Asp GlyAsp Ile Lys Thr Arg Gly Asp Phe 100 105 110 Arg Asp His Lys Val Ser ProArg Lys Ser Asp Ala Arg Trp Arg Thr 115 120 125 Ile Ile Gln Thr Ala ThrSer Lys Glu Glu Tyr Leu Asp Met Ile Lys 130 135 140 Glu Glu Phe Pro HisGlu Trp Ala Thr Lys Leu Gln Trp Leu Glu Tyr 145 150 155 160 Ser Ala AsnLys Leu Phe Pro Pro Gln Pro Glu Gln Tyr Val Ser Pro 165 170 175 Phe ThrGlu Ser Asp Leu Arg Cys His Glu Asp Leu His Asn Trp Arg 180 185 190 GluThr His Leu Tyr His Val Ser Ile Asp Ala Tyr Thr Phe Ile His 195 200 205Pro Val Ser Tyr Asp Gln Ala Gln Ser Asp Leu Glu Trp Met Ala Asp 210 215220 Leu Thr Arg Met Arg Glu Gly Leu Gly Ser Asp Thr Pro Ala Ser Thr 225230 235 240 Ser Ala Asp Gln Leu Val Pro Glu Arg Pro Pro Gly Leu Glu ValSer 245 250 255 Gly Asp Thr Thr Thr Gly Thr Gly Pro Ser Thr Ser Pro ThrThr Met 260 265 270 Asn Thr Pro Pro Ile Ile Ser Ser Thr Thr Ser Pro SerSer Ser Ser 275 280 285 His Cys Gly Ser Asn 290 4 143 PRT bean yellowdwarf virus 4 Asp Gly Arg Ser Asn Gln Asp Glu Gly Arg Thr Gly Val ArgHis Pro 1 5 10 15 Ser Leu Tyr Ile Cys Gly Pro Thr Arg Thr Gly Lys ThrThr Trp Ala 20 25 30 Arg Ser Leu Gly Arg His Asn Tyr Trp Asn Gly Thr IleAsp Phe Thr 35 40 45 Asn Tyr Asp Glu His Ala Thr Tyr Asn Ile Ile Asp AspIle Pro Phe 50 55 60 Lys Phe Val Pro Leu Trp Lys Gln Leu Ile Gly Cys GlnSer Asp Phe 65 70 75 80 Thr Val Asn Pro Lys Tyr Gly Lys Lys Lys Lys IleLys Gly Gly Ile 85 90 95 Pro Ser Ile Ile Leu Trp Asn Pro Asp Glu Asp TrpMet Leu Ser Met 100 105 110 Thr Ser Gln Gln Lys Asp Tyr Phe Glu Asp AsnCys Val Thr His Tyr 115 120 125 Met Cys Asp Gly Glu Thr Phe Phe Ala ArgGlu Ser Ser Ser His 130 135 140 5 1024 DNA bean yellow dwarf virus CDS(3)..(1010) 5 cc atg gga cct tct gct agc aag aac ttc aga ctc caa tct aaatat 47 Met Gly Pro Ser Ala Ser Lys Asn Phe Arg Leu Gln Ser Lys Tyr 1 510 15 gtt ttc ctt acc tac ccc aag tgc tca tct caa aga gat gat tta ttc 95Val Phe Leu Thr Tyr Pro Lys Cys Ser Ser Gln Arg Asp Asp Leu Phe 20 25 30cag ttt ctc tgg gag aaa ctc aca cct ttt ctt att ttc ttc ctt ggt 143 GlnPhe Leu Trp Glu Lys Leu Thr Pro Phe Leu Ile Phe Phe Leu Gly 35 40 45 gttgct tct gag ctt cat caa gat ggc act acc cac tat cat gct ctt 191 Val AlaSer Glu Leu His Gln Asp Gly Thr Thr His Tyr His Ala Leu 50 55 60 atc cagctt gat aaa aaa cct tgt att agg gat cct tct ttt ttc gat 239 Ile Gln LeuAsp Lys Lys Pro Cys Ile Arg Asp Pro Ser Phe Phe Asp 65 70 75 ttt gaa ggaaat cac cct aat atc cag cca gct aga aac tct aaa caa 287 Phe Glu Gly AsnHis Pro Asn Ile Gln Pro Ala Arg Asn Ser Lys Gln 80 85 90 95 gtc ctt gattac ata tca aag gac gga gat att aaa acc aga gga gat 335 Val Leu Asp TyrIle Ser Lys Asp Gly Asp Ile Lys Thr Arg Gly Asp 100 105 110 ttc cga gatcat aag gtc tct cct cgc aaa tct gac gca cga tgg cga 383 Phe Arg Asp HisLys Val Ser Pro Arg Lys Ser Asp Ala Arg Trp Arg 115 120 125 act att atccag act gca acg tct aag gag gag tat ctt gac atg atc 431 Thr Ile Ile GlnThr Ala Thr Ser Lys Glu Glu Tyr Leu Asp Met Ile 130 135 140 aaa gaa gaattc cct cat gaa tgg gca aca aag ctt caa tgg ctg gaa 479 Lys Glu Glu PhePro His Glu Trp Ala Thr Lys Leu Gln Trp Leu Glu 145 150 155 tat tca gccaac aaa tta ttt cct cca caa cct gag cag tac gtg tcg 527 Tyr Ser Ala AsnLys Leu Phe Pro Pro Gln Pro Glu Gln Tyr Val Ser 160 165 170 175 ccc ttcaca gaa tca gat ctc cgc tgc cac gaa gat ctg cac aac tgg 575 Pro Phe ThrGlu Ser Asp Leu Arg Cys His Glu Asp Leu His Asn Trp 180 185 190 aga gagacg cac cta tat cat gat gag gga agg act ggg gtc aga cac 623 Arg Glu ThrHis Leu Tyr His Asp Glu Gly Arg Thr Gly Val Arg His 195 200 205 ccc agcctc tac atc tgc gga cca act cgt acc gga aag acc acc tgg 671 Pro Ser LeuTyr Ile Cys Gly Pro Thr Arg Thr Gly Lys Thr Thr Trp 210 215 220 gct agaagt ctc ggg cga cac aac tac tgg aac ggg acc atc gac ttc 719 Ala Arg SerLeu Gly Arg His Asn Tyr Trp Asn Gly Thr Ile Asp Phe 225 230 235 acc aactac gat gaa cac gcc acc tat aat atc atc gac gac atc ccc 767 Thr Asn TyrAsp Glu His Ala Thr Tyr Asn Ile Ile Asp Asp Ile Pro 240 245 250 255 ttcaag ttc gtc cca ttg tgg aag caa tta ata ggt tgc cag tct gat 815 Phe LysPhe Val Pro Leu Trp Lys Gln Leu Ile Gly Cys Gln Ser Asp 260 265 270 ttcact gtc aac cct aaa tat gga aaa aag aag aaa ata aaa ggt ggg 863 Phe ThrVal Asn Pro Lys Tyr Gly Lys Lys Lys Lys Ile Lys Gly Gly 275 280 285 atccct tct ata att ctt tgg aat cct gac gaa gac tgg atg tta tca 911 Ile ProSer Ile Ile Leu Trp Asn Pro Asp Glu Asp Trp Met Leu Ser 290 295 300 atgaca agt caa cag aag gat tac ttt gaa gat aat tgc gtc acc cac 959 Met ThrSer Gln Gln Lys Asp Tyr Phe Glu Asp Asn Cys Val Thr His 305 310 315 tatatg tgt gac ggg gag act ttt ttt gct cgg gaa tcg tcg agt cac 1007 Tyr MetCys Asp Gly Glu Thr Phe Phe Ala Arg Glu Ser Ser Ser His 320 325 330 335tga acgtgcctga gctc 1024 6 335 PRT bean yellow dwarf virus 6 Met Gly ProSer Ala Ser Lys Asn Phe Arg Leu Gln Ser Lys Tyr Val 1 5 10 15 Phe LeuThr Tyr Pro Lys Cys Ser Ser Gln Arg Asp Asp Leu Phe Gln 20 25 30 Phe LeuTrp Glu Lys Leu Thr Pro Phe Leu Ile Phe Phe Leu Gly Val 35 40 45 Ala SerGlu Leu His Gln Asp Gly Thr Thr His Tyr His Ala Leu Ile 50 55 60 Gln LeuAsp Lys Lys Pro Cys Ile Arg Asp Pro Ser Phe Phe Asp Phe 65 70 75 80 GluGly Asn His Pro Asn Ile Gln Pro Ala Arg Asn Ser Lys Gln Val 85 90 95 LeuAsp Tyr Ile Ser Lys Asp Gly Asp Ile Lys Thr Arg Gly Asp Phe 100 105 110Arg Asp His Lys Val Ser Pro Arg Lys Ser Asp Ala Arg Trp Arg Thr 115 120125 Ile Ile Gln Thr Ala Thr Ser Lys Glu Glu Tyr Leu Asp Met Ile Lys 130135 140 Glu Glu Phe Pro His Glu Trp Ala Thr Lys Leu Gln Trp Leu Glu Tyr145 150 155 160 Ser Ala Asn Lys Leu Phe Pro Pro Gln Pro Glu Gln Tyr ValSer Pro 165 170 175 Phe Thr Glu Ser Asp Leu Arg Cys His Glu Asp Leu HisAsn Trp Arg 180 185 190 Glu Thr His Leu Tyr His Asp Glu Gly Arg Thr GlyVal Arg His Pro 195 200 205 Ser Leu Tyr Ile Cys Gly Pro Thr Arg Thr GlyLys Thr Thr Trp Ala 210 215 220 Arg Ser Leu Gly Arg His Asn Tyr Trp AsnGly Thr Ile Asp Phe Thr 225 230 235 240 Asn Tyr Asp Glu His Ala Thr TyrAsn Ile Ile Asp Asp Ile Pro Phe 245 250 255 Lys Phe Val Pro Leu Trp LysGln Leu Ile Gly Cys Gln Ser Asp Phe 260 265 270 Thr Val Asn Pro Lys TyrGly Lys Lys Lys Lys Ile Lys Gly Gly Ile 275 280 285 Pro Ser Ile Ile LeuTrp Asn Pro Asp Glu Asp Trp Met Leu Ser Met 290 295 300 Thr Ser Gln GlnLys Asp Tyr Phe Glu Asp Asn Cys Val Thr His Tyr 305 310 315 320 Met CysAsp Gly Glu Thr Phe Phe Ala Arg Glu Ser Ser Ser His 325 330 335 7 815DNA bean yellow dwarf virus CDS (3)..(803) 7 cc atg gac aag agg ctc ttcatc tcc cat gtg atc ctc atc ttt gca 47 Met Asp Lys Arg Leu Phe Ile SerHis Val Ile Leu Ile Phe Ala 1 5 10 15 ctc atc ttg gtg atc tct acc cccaat gtg ttg gca gag agc caa cca 95 Leu Ile Leu Val Ile Ser Thr Pro AsnVal Leu Ala Glu Ser Gln Pro 20 25 30 gac cct aag cca gat gag ttg cat aagagc agc aag ttc act ggt ctc 143 Asp Pro Lys Pro Asp Glu Leu His Lys SerSer Lys Phe Thr Gly Leu 35 40 45 atg gag aac atg aag gtg ctc tat gat gacaac cat gtg tca gca atc 191 Met Glu Asn Met Lys Val Leu Tyr Asp Asp AsnHis Val Ser Ala Ile 50 55 60 aat gtg aag tct att gac caa tcc ctc tac tttgac ctc atc tac tct 239 Asn Val Lys Ser Ile Asp Gln Ser Leu Tyr Phe AspLeu Ile Tyr Ser 65 70 75 atc aag gac act aag ttg gga aac tat gac aat gtgagg gtg gag ttc 287 Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn Val ArgVal Glu Phe 80 85 90 95 aag aac aag gac ttg gct gac aag tac aag gac aagtat gtg gat gtg 335 Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys TyrVal Asp Val 100 105 110 ttt gga gct aac tac tat tac caa tgc tac ttc tctaag aaa acc aat 383 Phe Gly Ala Asn Tyr Tyr Tyr Gln Cys Tyr Phe Ser LysLys Thr Asn 115 120 125 gac atc aac agc cac caa act gac aag aga aag acttgc atg tat ggt 431 Asp Ile Asn Ser His Gln Thr Asp Lys Arg Lys Thr CysMet Tyr Gly 130 135 140 ggt gtg act gag cac aac gga aac caa ttg gac aaatac agg agc atc 479 Gly Val Thr Glu His Asn Gly Asn Gln Leu Asp Lys TyrArg Ser Ile 145 150 155 act gtg agg gtg ttt gag gat ggt aag aac ctc ctctct ttt gat gtg 527 Thr Val Arg Val Phe Glu Asp Gly Lys Asn Leu Leu SerPhe Asp Val 160 165 170 175 caa act aac aag aag aag gtg act gct caa gagttg gac tac ctc act 575 Gln Thr Asn Lys Lys Lys Val Thr Ala Gln Glu LeuAsp Tyr Leu Thr 180 185 190 agg cac tac ttg gtg aag aac aag aag ctc tatgag ttc aac aac agc 623 Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr GluPhe Asn Asn Ser 195 200 205 cct tat gag act gga tac atc aag ttc att gagaat gag aac agc ttc 671 Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu AsnGlu Asn Ser Phe 210 215 220 tgg tat gac atg atg cct gca cca gga gac aagttt gac caa tct aag 719 Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys PheAsp Gln Ser Lys 225 230 235 tac ctc atg atg tac aat gac aac aag atg gtggac tct aag gat gtg 767 Tyr Leu Met Met Tyr Asn Asp Asn Lys Met Val AspSer Lys Asp Val 240 245 250 255 aag att gag gtg tac ctt acc acc aag aagaag taa gtcttcgagc tc 815 Lys Ile Glu Val Tyr Leu Thr Thr Lys Lys Lys260 265 8 266 PRT bean yellow dwarf virus 8 Met Asp Lys Arg Leu Phe IleSer His Val Ile Leu Ile Phe Ala Leu 1 5 10 15 Ile Leu Val Ile Ser ThrPro Asn Val Leu Ala Glu Ser Gln Pro Asp 20 25 30 Pro Lys Pro Asp Glu LeuHis Lys Ser Ser Lys Phe Thr Gly Leu Met 35 40 45 Glu Asn Met Lys Val LeuTyr Asp Asp Asn His Val Ser Ala Ile Asn 50 55 60 Val Lys Ser Ile Asp GlnSer Leu Tyr Phe Asp Leu Ile Tyr Ser Ile 65 70 75 80 Lys Asp Thr Lys LeuGly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys 85 90 95 Asn Lys Asp Leu AlaAsp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe 100 105 110 Gly Ala Asn TyrTyr Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn Asp 115 120 125 Ile Asn SerHis Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly 130 135 140 Val ThrGlu His Asn Gly Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr 145 150 155 160Val Arg Val Phe Glu Asp Gly Lys Asn Leu Leu Ser Phe Asp Val Gln 165 170175 Thr Asn Lys Lys Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu Thr Arg 180185 190 His Tyr Leu Val Lys Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser Pro195 200 205 Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu Asn Ser PheTrp 210 215 220 Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln SerLys Tyr 225 230 235 240 Leu Met Met Tyr Asn Asp Asn Lys Met Val Asp SerLys Asp Val Lys 245 250 255 Ile Glu Val Tyr Leu Thr Thr Lys Lys Lys 260265 9 4 PRT Artificial Sequence Description of ArtificialSequencePeptide signal which targets nascent protein to the endoplasmicreticulum. 9 Lys Asp Glu Leu 1 10 6 PRT Artificial Sequence Descriptionof Artificial SequencePeptide signal which targets nascent protein tothe endoplasmic reticulum. 10 Ser Glu Lys Asp Glu Leu 1 5 11 16 DNAArtificial Sequence Description of Artificial SequencePrimer foramplifying a GFP expression cassette. 11 agctcgacca ggatgg 16 12 16 DNAArtificial Sequence Description of Artificial SequencePrimer foramplifying a GFP expression cassette. 12 gtcctgctgg agttcg 16 13 30 DNAArtificial Sequence Description of Artificial SequencePrimer foramplifying SIR of BYDV. 13 ggaattccga gtgtacttca agtcagttgg 30 14 32 DNAArtificial Sequence Description of Artificial SequencePrimer foramplifying SIR of BYDV. 14 ggaagcttgg gatcccttct ataattcttt gg 32 15 30DNA Artificial Sequence Description of Artificial SequencePrimer foramplifying LIR of BYDV. 15 tggcgcgccg ctctagcaga aggcatgttg 30 16 32 DNAArtificial Sequence Description of Artificial SequencePrimer foramplifying LIR of BYDV. 16 ttggccggcc gtacgaataa ttcgtatccg ac 32 17 28DNA Artificial Sequence Description of Artificial SequenceLinker forreplacing plasmid multiple cloning region. 17 agctggcgcg ccgtttaaacggccggcc 28 18 28 DNA Artificial Sequence Description of ArtificialSequenceLinker for replacing plasmid multiple cloning region. 18ttaaccggcc ggcaaatttg ccgcgcgg 28 19 20 DNA Artificial SequenceDescription of Artificial SequencePrimer for amplifying a form of theBYDV Rep gene. 19 cggataacaa tttcacacag 20 20 18 DNA Artificial SequenceDescription of Artificial SequencePrimer for amplifying a form of theBYDV Rep gene. 20 ctcagctaat taagctta 18 21 27 DNA Artificial SequenceDescription of Artificial SequencePrimer for mutagenesis of Rep C1 gene.21 ccttgtatta gggtccttct tttttcg 27 22 27 DNA Artificial SequenceDescription of Artificial SequencePrimer for mutagenesis of Rep C1 gene.22 cgaaaaaaga aggaccctaa tacaagg 27

1. A pair of recombinant nucleic acid molecules wherein a first moleculecomprises at least a portion of a long intergenic region (LIR) of ageminivirus genome, wherein said first molecule lacks a functionalgeminiviral coat protein encoding sequence; and a second moleculecomprising a geminiviral replicase gene operably linked to a fruitripening-dependent promoter.
 2. The pair of recombinant nucleic acidmolecules of claim 1 wherein said first molecule further comprises anSIR.
 3. The pair of recombinant nucleic acid molecules of claim 1,wherein said first molecule further comprises a plant-functionalpromoter.
 4. The pair of recombinant nucleic acid molecules of claim 3,wherein said plant-functional promoter is selected from the groupconsisting of CAMV 35S, tomato E8, patatin, ubiquitin, mannopinesynthase (mas), rice actin 1, soybean seed protein glycinin (Gy1) andsoybean vegetative storage protein (vsp).
 5. The pair of recombinantnucleic acid molecules of claim 1, wherein said first molecule furthercomprises a gene of interest.
 6. The pair of recombinant nucleic acidmolecules of claim 1, wherein said gene of interest of said firstmolecule is a heterologous gene.
 7. The pair of recombinant nucleic acidmolecules of claim 1, wherein said gene of interest of said first DNAmolecule is selected from the group consisting of a gene encodingluciferase, glucuronosidase (GUS), green fluorescent protein (GFP),shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), E. coli labiletoxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis Bsurface antigen (HBsAg).
 8. The pair of recombinant nucleic acidmolecules of claim 1, wherein said first molecule further comprises aplant-functional termination sequence.
 9. The pair of recombinantnucleic acid molecules of claim 8, wherein said plant-functionaltermination sequence is selected from the group consisting of nopalinesynthase (nos), vegetative storage protein (vsp), pin2, and geminiviralshort intergenic (sir) termination sequences.
 10. The pair ofrecombinant nucleic acid molecules of claim 1 wherein said firstmolecule is single stranded.
 11. A recombinant nucleic acid moleculecomprising at least a portion of a long intergenic region (LIR) of ageminivirus genome, and a geminiviral replicase gene operably linked toa fruit ripening-dependent promoter.
 12. The recombinant nucleic acidmolecule of claim 11 further comprising an SIR.
 13. The recombinantnucleic acid molecule of claim 11 further comprising a plant-functionalpromoter.
 14. The recombinant nucleic acid molecule of claim 13, whereinsaid plant-functional promoter is selected from the group consisting ofCaMV 35S, tomato E8, patatin, ubiquitin, mannopine synthase (mas), riceactin 1, soybean seed protein glycinin (Gy1) and soybean vegetativestorage protein (vsp).
 15. The recombinant nucleic acid molecule ofclaim 11 further comprising a gene of interest.
 16. The recombinantnucleic acid molecule of claim 11, wherein said gene is a heterologousgene.
 17. The recombinant nucleic acid molecule of claim 11, whereinsaid gene of interest of said first DNA construct is selected from thegroup consisting of a gene encoding luciferase, glucuronosidase (GUS),green fluorescent protein (GFP), shigatoxin B (StxB), staphylococcusenterotoxin B (SEB), E. coli labile toxin B (LT-B), Norwalk virus capsidprotein (NVCP), and hepatitis B surface antigen (HbsAg).
 18. Therecombinant nucleic acid molecule of claim 11, further comprising aplant-functional termination sequence.
 19. The recombinant nucleic acidmolecule of claim 18, wherein said plant-functional termination sequenceis selected from the group consisting of nopaline synthase (nos),vegetative storage protein (vsp), pin2, and geminiviral short intergenic(sir) termination sequences.
 20. The recombinant nucleic acid moleculeof claim 11, wherein said nucleotide sequence is optimized forexpression in plants by having at least one codon degenerate to acorresponding codon of the native protein encoding sequence.
 21. Therecombinant nucleic acid molecule of claim 11, which is single stranded.22. An expression vector comprising a selectable marker gene and atleast a portion of a long intergenic region (LIR) of a geminivirusgenome, a restriction site for insertion of a gene of interest, and afunctional geminiviral replicase gene operably linked to a fruitripening-dependent promoter and wherein said nucleic acid sequence lacksa functional geminiviral coat protein encoding sequence.
 23. The vectorof claim 22, further comprising a gene of interest.
 24. The vector ofclaim 22, wherein the gene is a heterologous gene.
 25. The vector ofclaim 22, further comprising an SIR.
 26. The vector of claim 22, whichlacks a functional geminiviral replicase gene.
 27. The vector of claim22, wherein said gene of interest is flanked by two of said LIRportions.
 28. The vector of claim 22, wherein the 5′ end of said gene ofinterest is operably linked to a plant-functional promoter sequence. 29.The vector of claim 22, wherein said gene of interest is selected fromthe group consisting of a gene encoding luciferase, glucuronosidase(GUS), green fluorescent protein (GFP), shigatoxin B (StxB),staphylococcus enterotoxin B (SEB), labile toxin B (LT-B), Norwalk viruscapsid protein (NVCP), and hepatitis B surface antigen (HbsAg).
 30. Thevector of claim 22, wherein the 3′ end of said gene is operably linkedto a plant-functional termination sequence.
 31. The vector of claim 22,further comprising an E. coli origin of replication.
 32. The vector ofclaim 31, further comprising an Agrobacterium tumefaciens origin ofreplication.
 33. The vector of claim 25, wherein said nucleotidesequence is flanked by left and right T-DNA border regions ofAgrobacterium tumefaciens.
 34. An expression vector comprising aselectable marker gene and at least a portion of a geminivirus genome, arestriction site for insertion of a gene of interest, wherein saidexpression vector lacks a functional geminviral coat protein encodingsequence.
 35. A strain of E. coli transfected with the expression vectorof claim
 31. 36. A strain of Agrobacterium tumefaciens transfected withthe expression vector of claim
 32. 37. The strain of claim 36, furthercomprising a helper tumor-inducing (Ti) plasmid.
 38. A transgenic plantcell transformed with a nucleic acid having at least a portion of a longintergenic region (LIR) of a geminivirus genome, a gene of interest,wherein said nucleic acid lacks a functional geminiviral coat proteinencoding sequence.
 39. A transgenic plant cell transformed with anucleic acid comprising at least a portion of a long intergenic region(LIR) of a geminivirus genome, a restriction site for of insertion of agene of interest, and a functional geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter and wherein said nucleicacid sequence lacks a functional geminiviral coat protein encodingsequence.
 40. The transgenic plant cell of claim 38, further comprisinga heterologous gene.
 41. The transgenic plant cell of claim 38, whereinsaid nucleic acid lacks a functional geminiviral replicase gene.
 42. Thetransgenic plant cell of claim 38, wherein the nucleic acid is presentin nuclear episomes in the cell.
 43. The transgenic plant cell of claim38, wherein the 5′ end of said gene of interest is operably linked to aplant-functional promoter sequence.
 44. The transgenic plant cell ofclaim 38, wherein said gene of interest is selected from the groupconsisting of a gene encoding luciferase, glucuronosidase (GUS), greenfluorescent protein (GFP), shigatoxin B (StxB), staphylococcusenterotoxin B (SEB), labile toxin B (LT-B), Norwalk virus capsid protein(NVCP), and hepatitis B surface antigen (HBsAg).
 45. The transgenicplant cell of claim 38, wherein the 3 ′ end of said gene of interest isoperably linked to a plant-functional termination sequence.
 46. Thetransgenic plant cell of claim 38, further comprising a viral replicaseencoding sequence operably linked to a plant functional promoter and atermination sequence.
 47. The transgenic plant cell of claim 38, whereintranscription of the viral replicase encoding sequence is regulated byan inducible promoter.
 48. The transgenic plant cell of claim 38,wherein the 5′ end of the viral replicase encoding sequence is operablylinked to a tissue-specific promoter.
 49. The transgenic plant cell ofclaim 48, wherein the tissue-specific promoter is selected from thegroup consisting of glucocorticoid, estrogen, jasmonic acid, insecticideRH5992, copper, tetracycline, and alcohol-inducible promoters.
 50. Thetransgenic plant cell of claim 46, wherein the viral replicase encodingsequence encodes a wild-type geminiviral replicase.
 51. The transgenicplant cell of claim 46, wherein the viral replicase encoding sequenceprovided as an expression cassette or viral replicon.
 52. A transgenicplant seed transformed with a nucleic acid having at least a portion ofa long intergenic region (LIR) of a geminivirus genome, a gene ofinterest, wherein said nucleic acid lacks a functional geminiviral coatprotein encoding sequence.
 53. A transgenic plant seed transformed witha nucleic acid comprising at least a portion of a long intergenic region(LIR) of a geminivirus genome, a restriction site for insertion of agene of interest, and a functional geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter and wherein said nucleicacid sequence lacks a functional geminiviral coat protein encodingsequence.
 54. The seed of claim 52, further comprising a heterologousgene.
 55. The seed of claim 54, wherein said nucleic acid lacks afunctional geminiviral replicase gene.
 56. The seed of claim 54, furthercomprising a viral replicase encoding sequence expressed in trans withsaid nucleotide sequence.
 57. The seed of claim 56, wherein the 5′ endof the viral replicase encoding sequence is operably linked to atissue-specific promoter.
 58. The seed of claim 54, which is selectedfrom tobacco, tomato, potato, banana, soybean, pepper, wheat, rye, rice,spinach, carrot, maize and corn.
 59. A method of transforming a plantcell comprising contacting the plant cell with a strain of Agrobacteriumtumefaciens as in claim 36 under conditions effective to transfer andintegrate said nucleotide sequence into the nuclear genome of the cell.60. A method of producing a transgenic plant comprising transforming aplant cell as in claim 59 and regenerating the plant cell.
 61. A methodof transforming a plant cell comprising subjecting the plant cell tomicroparticle bombardment with solid particles loaded with a DNAconstruct as in claim 1 under conditions effective to transfer andintegrate said nucleotide sequence into the nuclear genome of the cell.62. A method of producing a transgenic plant comprising transforming aplant cell as in claim 61 and regenerating the plant cell.
 63. A methodof amplifying a heterologous nucleotide sequence in a transgenic plantcomprising subjecting the transgenic plant of claim 60 to a wild-typegeminivirus, which expresses a viral replicase in planta that rescuesand replicates said nucleotide sequence in cells of said plant.
 64. Amethod of overproducing a protein in a plant comprising subjecting thetransgenic plant of claim 60 to a wild-type geminivirus, which expressesa viral replicase in planta that rescues and replicates said nucleotidesequence in said plant.
 65. A method of amplifying a heterologousnucleotide sequence in a transgenic plant comprising subjecting thetransgenic plant of claim 60 to a chemical or developmental agent, whichinduces expression of a viral replicase in planta that rescues andreplicates said nucleotide sequence in said plant.
 66. The method ofclaim 65, wherein said inducible promoter is selected from the groupconsisting of glucocorticoid, estrogen, and alcohol-inducible promoters.67. The method of claim 65, wherein replication of said viral replicaseis expressed in trans with said nucleotide sequence.
 68. A method ofoverproducing a protein in a plant, comprising subjecting the transgenicplant of claim 60 to a chemical or developmental agent, which inducesexpression of a viral replicase in planta that rescues and replicatessaid nucleotide sequence in said plant.
 69. A recombinant nucleic acidmolecule comprising a functional geminiviral replicase gene operablylinked to a fruit ripening-dependent promoter.
 70. A vector comprising afunctional geminiviral replicase gene operably linked to a fruitripening-dependent promoter.
 71. A transgenic plant cell transformedwith a nucleic acid comprising a functional geminiviral replicase geneoperably linked to a fruit ripening-dependent promoter.
 72. A transgenicplant seed transformed with a nucleic acid comprising a functionalgeminiviral replicase gene operably linked to a fruit ripening-dependentpromoter.